Multiparametric fluorescence in situ hybridization

ABSTRACT

The invention relates to a set of combinatorially labeled oligonucleotide probes each member thereof: (i) having a predetermined label distinguishable from the label of any other member of said set, and (ii) being capable of specifically hybridizing with a predetermined chromosome or nucleic acid molecule, and to the use of such molecules, alone or in concert with nucleic acid amplification methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 08/640,657 (filed May 1, 1996), which is acontinuation in part of U.S. patent application Ser. No. 08/580,717(filed Dec. 29, 1995), which is a continuation in part of U.S. patentapplication Ser. No. 08/577,622 (filed Dec. 22, 1995), all of whichapplications are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to nucleic acid chemistry, and morespecifically to reagents and methods for accomplishing multiplex imageanalysis of chromosomes and chromosomal fragments. The invention may beused to diagnose chromosomal abnormalities, infectious agents, etc. Thisinvention was made in part using Government funds. The Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The determination of the presence and condition of chromosomesand chromosomal fragments in a biological sample is of immenseimportance in the diagnosis of disease. Traditionally, suchdeterminations have been done manually by inspecting metaphasechromosomal preparations that have been treated with specialized stainsto reveal characteristic banding patterns. Unfortunately, theinterpretation of such banding patterns requires substantial skill andis technically difficult. Hence, alternate methods of analyzingchromosomal presence and arrangement have been sought.

[0004] One alternative approach to the problem of chromosomeidentification has involved the use of labeled chromosome-specificoligonucleotide probes to label repetitive sequences of interphasechromosomes (Cremer, T. et al., Hum. Genet. 74:346-352 (1986); Cremer,T. et al., Exper. Cell Res. 176:119-220 (1988)). Such methods have beenshown to be useful in the prenatal diagnosis of Down's Syndrome, as wellas in the detection of chromosomal abnormalities associated with tumorcell lines. Chromosome-specific probes of repetitive DNA that localizeto discrete sub-regions of a chromosome are, however, unsuitable foranalyses of many types of chromosomal abnormalities (e.g.,translocations or deletions).

[0005] Ward, D. C. et al. (PCT Application WO/05789, herein incorporatedby reference) discloses a chromosomal in situ suppression (“CISS”)hybridization method for specifically labeling selected mammalianchromosomes in a manner that permits the recognition of chromosomalaberrations. In that method, sample DNA is denatured and permitted tohybridize with a mixture of fluorescently labeled chromosome-specificprobes having high genetic complexity and unlabeled non-specificcompetitor probes. Chromosomal images were obtained as described byManuelidis, L. et al. (Chromosoma 96:397-410 (1988), herein incorporatedby reference). The method provides a rapid and highly specificassessment of individual mammalian chromosomes. The method permits, byjudicious selection of appropriate probes and/or labels, thevisualization of sub-regions of some or all of the chromosomes in apreparation. For example, by using more than one probe, each specificfor a sub-region of a target chromosome, the method permits thesimultaneous analysis of several sub-regions on that chromosome. Thenumber of available fluorophores limits the number of chromosomes orchromosomal sub-regions that can be simultaneously visualized.

[0006] As described in PCT Application WO/05789, a “combinatorial”variation of the CISS method can be employed. In the simplest case, twofluors permit three different chromosomes or chromosomal sub-regions tobe simultaneously visualized. In this variation, a hybridization probemixture is made from a single set of probe sequences composed of twohalves, each separately labeled with a different fluorophore. Uponhybridization, the two fluorophores produce a third fluorescence signalthat is optically distinguishable from the color of the individualfluorophores. Extension of this approach to Boolean combinations of nfluorophores permits the labeling of 2^(n)-1 chromosomes.

[0007] Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392(1992), herein incorporated by reference) describes the use of anepi-fluorescent microscope equipped with a digital imaging camera andcomputer software to “pseudocolor” the fluorescence patterns obtainedfrom simultaneous in situ hybridization with seven probes using threefluorophores. The use of wavelength-selective filters allows one toisolate and collect separate gray scale images of each fluorophore.These images can be subsequently merged via appropriate software. Thesensitivity and linearity of CCD cameras surmounts the technicaldifficulties inherent in color film-based photomicroscopy.

[0008] Although such efforts have increased the number of chromosomesthat can be simultaneously detected and analyzed using in situhybridization methods, it would be highly desirable to define a set offluorophores having distinguishable emission spectra to permit thesimultaneous detection and analysis of large numbers of differentchromosomes and chromosomal sub-regions. The present invention providessuch reagents as well as methods and apparatus for their use.

SUMMARY OF THE INVENTION

[0009] The invention concerns reagents and methods for combinatoriallabeling of nucleic acid probes sufficient to permit the visualizationand simultaneous identification of all 22 autosomal human chromosomesand the human X and Y chromosomes, or defined sub-regions thereof. Suchspecific labeling of entire chromosomes or defined sub-regions thereofis referred to as “painting.” The invention further concerns reagentsand methods for combinatorial labeling of nucleic acid probes sufficientto permit the characterization of bacteria, viruses and/or lowereukaryotes that may be present in a clinical or non-clinicalpreparation.

[0010] In detail, the invention concerns a set of combinatoriallylabeled oligonucleotide probes comprised of a first and a second subsetof probes, wherein:

[0011] (A) each member of the first subset of probes comprises aplurality of an oligonucleotide: (i) being linked or coupled to apredetermined label distinguishable from the label of any other memberof the first or second subsets of probes, and (ii) being capable ofspecifically hybridizing with one predetermined autosomal or sexchromosome of a human karyotype;

[0012] the first subset of probes set having sufficient members to becapable of specifically hybridizing each autosomal or sex chromosome ofthe human karyotype to at least one member, and

[0013] (B) each member of the second subset of probes comprises aplurality of an oligonucleotide: (i) being linked or coupled to apredetermined label distinguishable from the label of any other memberof the first or second subset, and (ii) being capable of specificallyhybridizing with one extra-chromosomal polynucleotide copy of apredetermined region of an autosomal or sex chromosome of the humankaryotype.

[0014] The invention further concerns a set of combinatorially labeledoligonucleotide probes comprised of a first subset of genotypic probesand a second subset of phenotypic probes, wherein:

[0015] (A) each member of the first subset of genotypic probes comprisesa plurality of an oligonucleotide: (i) being linked or coupled to apredetermined label distinguishable from the label of any other memberof the first or second subsets of probes, and (ii) being capable ofspecifically hybridizing with a region of a nucleic acid of apreselected bacterium, virus or lower eukaryote;

[0016] the first subset of probes set having sufficient members to becapable of distinguishing the preselected bacterium, virus, or lowereukaryote from other bacteria, viruses, or lower eukaryotes; and

[0017] (B) each member of the second subset of phenotypic probescomprises a plurality of an oligonucleotide: (i) being linked or coupledto a predetermined label distinguishable from the label of any othermember of the first or second subset, and (ii) being capable ofspecifically hybridizing with a predetermined polynucleotide region ofthe chromosome of the preselected bacterium, virus, or lower eukaryote,or an extra-chromosomal copy thereof so as to permit the determinationof whether the preselected bacterium, virus, or lower eukaryote exhibitsa preselected phenotype.

[0018] The invention additionally concerns a method of simultaneouslyidentifying and distinguishing the individual autosomal and sexchromosomes of a human karyotype which comprises the steps:

[0019] (I) contacting a preparation of the chromosomes, insingle-stranded form, under conditions sufficient to permit nucleic acidhybridization to occur with a set of combinatorially labeledoligonucleotide probes comprised of a first and a second subset ofprobes, wherein:

[0020] (A) each member of the first subset of probes comprises aplurality of an oligonucleotide: (i) being linked or coupled to apredetermined label distinguishable from the label of any other memberof the first or second subsets of probes, and (ii) being capable ofspecifically hybridizing with one predetermined autosomal or sexchromosome of a human karyotype;

[0021] the first subset of probes set having sufficient members to becapable of specifically hybridizing each autosomal or sex chromosome ofthe human karyotype to at least one member; and

[0022] (B) each member of the second subset of probes comprises aplurality of an oligonucleotide: (i) being linked or coupled to apredetermined label distinguishable from the label of any other memberof the first or second subset, and (ii) being capable of specificallyhybridizing with an a predetermined extra-chromosomal polynucleotidecopy of a region of an autosomal or sex chromosome of the humankaryotype.

[0023] (II) for each chromosome of the preparation hybridized to amember of the first subset of probes, detecting and identifying thepredetermined label of that member and correlating the identity of thelabel of that member with the identity of the autosomal or sexchromosome of the human karyotype with which that member specificallyhybridizes, to thereby identify the chromosome hybridized to the member,

[0024] (III) repeating step (II) until each autosomal and sex chromosomeof the human karyotype has been identified in the preparation

[0025] (IV) for each member of the second subset of probes hybridized toa predetermined extra-chromosomal polynucleotide copy of a region of anautosomal or sex chromosome detecting and identifying the predeterminedlabel of that member and correlating the identity of the label of thatmember with the identity of the region of the autosomal or sexchromosome of the human karyotype with which that member specificallyhybridizes, to thereby identify the region of the autosomal or sexchromosome hybridized to the member;

[0026] (V) repeating step (IV) for each member of the second subset ofprobes.

[0027] The invention additionally concerns a method of simultaneouslyidentifying and distinguishing a preselected bacterium, virus, or lowereukaryote from other bacteria, viruses or lower eukaryotes that may bepresent in a sample which comprises the steps:

[0028] (I) contacting a preparation suspected to contain the preselectedbacterium, virus, or lower eukaryote, under conditions sufficient topermit in situ nucleic acid hybridization to occur, with a set ofcombinatorially labeled oligonucleotide probes comprised of a firstsubset of genotypic probes and a second subset of phenotypic probes,wherein:

[0029] (A) each member of the first subset of genotypic probes comprisesa plurality of an oligonucleotide: (i) being linked or coupled to apredetermined label distinguishable from the label of any other memberof the first or second subsets of probes, and (ii) being capable ofspecifically hybridizing with a region of a nucleic acid of thepreselected bacterium, virus or lower eukaryote;

[0030] the first subset of probes set having sufficient members to becapable of distinguishing the preselected bacterium, virus, or lowereukaryote from other bacteria, viruses, or lower eukaryotes present inthe preparation; and

[0031] (B) each member of the second subset of probes comprises aplurality of an oligonucleotide: (i) being linked or coupled to apredetermined label distinguishable from the label of any other memberof the first or second subset, and (ii) being capable of specificallyhybridizing with a predetermined polynucleotide region of the nucleicacid of the preselected bacterium, virus, or lower eukaryote, or anextra-chromosomal copy thereof so as to permit the determination ofwhether the preselected bacterium, virus, or lower eukaryote exhibits apreselected phenotype.

[0032] (II) for each member of the first subset of probes hybridized toa region of a chromosome of a preselected bacterium, virus or lowereukaryote, detecting and identifying the predetermined label of thatmember and correlating the identity of the label of that member with theidentity of the bacterium, virus or lower eukaryote with which thatmember specifically hybridizes, to thereby identify the bacterium, virusor lower eukaryote hybridized to the member;

[0033] (III) repeating step (II) for each member of the first subset ofprobes;

[0034] (IV) for each member of the second subset of probes hybridized toa predetermined polynucleotide region of the chromosome of thepreselected bacterium, virus, or lower eukaryote, or anextra-chromosomal copy thereof, detecting and identifying thepredetermined label of that member and correlating the identity of thelabel of that member with the identity of the predetermined region, tothereby identify the presence of the predetermined region on achromosome of the preselected bacteria, virus or lower eukaryote;

[0035] (V) repeating step (IV) for each member of the second subset ofprobes.

[0036] The invention particularly contemplates the embodiments in whichthe members of the above sets of probes are detectably labeled withfluorophores, and, wherein at least one member of the set iscombinatorially labeled with either one, two, three, four or fivefluorophores selected from the group consisting of the fluorophoresFITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of the setis labeled with at least one fluorophore selected from the fluorophoregroup.

[0037] The invention additionally provides a set of combinatoriallylabeled oligonucleotide probes, each member thereof: (i) having apredetermined label distinguishable from the label of any other memberof the set, and (ii) being capable of specifically hybridizing with atelomeric region of one predetermined autosomal or sex chromosome of ahuman karyotype; the set having sufficient members to be capable ofspecifically hybridizing each autosomal or sex chromosome of the humankaryotype to at least one member.

[0038] The invention additionally provides a method of simultaneouslyidentifying and distinguishing the individual autosomal and sexchromosomes of a human karyotype which comprises the steps:

[0039] (a) contacting a preparation of the chromosomes, insingle-stranded form, under conditions sufficient to permit nucleic acidhybridization to occur with a set of combinatorially labeledoligonucleotide probes, each member thereof: (i) having a predeterminedlabel distinguishable from the label of any other member of the set, and(ii) being capable of specifically hybridizing with a telomeric regionof one predetermined autosomal or sex chromosome of a human karyotype;the set having sufficient members to be capable of specificallyhybridizing each autosomal or sex chromosome of the human karyotype toat least one member, wherein the contacting thereby causes at least oneof each autosomal or sex chromosome of the preparation to becomehybridized to at least one member of the set of probes;

[0040] (b) for each chromosome of the preparation hybridized to a memberof the set of probes, detecting and identifying the predetermined labelof that member and correlating the identity of the label of that memberwith the identity of the autosomal or sex chromosome of the humankaryotype with which that member specifically hybridizes, to therebyidentify the chromosome hybridized to the member, and

[0041] (c) repeating step (b) until each autosomal and sex chromosome ofthe human karyotype has been identified in the preparation.

BRIEF DESCRIPTION OF THE FIGURES

[0042]FIG. 1 provides a schematic illustration of a CCD camera andmicroscope employed in accordance with the present methods.

[0043]FIG. 2 shows the raw data from a karyotypic analysis ofchromosomes from a bone marrow patient (BM2486). Adjacent to each sourceimage is a chromosome “mask” generated by the software program. In FIG.2, panels A and B are the DAPI image and mask; panels C and D are FITCimage and mask; panels E and F are Cy3 image and mask; panels G and Hare Cy3.5 image and mask; panels I and J are Cy5 image and mask; andpanels K and L are Cy7 image and mask.

[0044]FIGS. 3A and 3B show the identification of individual chromosomesby spectral signature of patient BM2486. FIG. 2 is the same photographas FIG. 3A, except that it is gray scale pseudocolored. FIG. 3B displaysthe karyotypic array of the chromosomes.

[0045]FIG. 4 shows the differentiation of bacteria by in situhybridization. Panels A-E represent in situ hybridization assay resultson a laboratory derived mixture of three bacteria (F. nucleatum, A.actinomycetemcomitans and E. corrodens) using a mixture of genomic DNAprobes for each organism. Panel A shows all the bacteria present in themicroscope field detected by (DAPI), a general DNA binding fluorophore.Panel B shows F. nucleatum using Cascade Blue detection. Panel C showsA. actinomycetemcomitans using FITC detection. Panel D shows E.corrodens using Rhodamine detection. Panel E is a computer-mergedcomposite of panels B-D showing the differentiation of each bacteria inthe sample. Panel F is a similar analysis of a mixture of C. gingivalis(using rhodamine detection) and P. intermedia (using FITC detection)when hybridized with a combination of genomic DNA probes for thoseorganisms. Panel G shows an in situ hybridization assay for acombination of seven different bacteria hybridized with a mixture ofseven genomic DNA probes. In panel G, the numbers (1)-(7) identify thebacteria. F. nucleatum (1), E. corrodens (2) and A.actinomycetemcomitans (3) are shown in blue (Cascade Blue detection). P.gingivalis (4) and C. ochracea (5) are shown in red (Rhodaminedetection), whereas P. intermedia (6) and C. gingivalis (7) are seen inyellow (FITC detection). Panel H represents an in situ hybridizationassay for the presence of A. actinomycetemcomitans (using FITCdetection) in a plaque sample obtained from a patient with localizedjuvenile periodontist.

[0046]FIG. 5A shows a normal male metaphase chromosomal spread afterhybridization with a 24-color set of telomere-specific probes, shown asa pseudocolorized image. FIG. 5B shows the final karyotype generated onthe basis of the boolean spectral signature of the telomere-specificprobes.

[0047]FIG. 6A shows the hybridization pattern of the chromosome 8subtelomeric YACs (telomere-specific probes) on a normal metaphasechromosomal pread. FIG. 6B shows the hybridization pattern of the sameprobes (as those used in FIG. 6A) on a metaphase chromosomal spread froma patient with a myeloproliferative disorder. Previous cytogeneticanalysis of this patient using G-banding revealed a trisomy 8 as theonly change; the M-FISH telomere-specific probes show a split telomeresignal on the short arms of two of the three chromosome 8, indicating anadditional change: an inversion in this region.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] A. Overview of the Invention

[0049] Fluorescence in situ hybridization (FISH) is used in a variety ofareas of research and clinical diagnostics (Gray, J. W. et al., CurrOpin Biotech 3:623-631 (1992); Xing, Y. et al., In: The Causes andConsequences of Chromosomal Aberrations. I. R. Kirsch Ed. CRC Press,Boca Raton, pages 3-28 (1993)). For the study of the chromosomal andsuprachromosomal organization of the cell nucleus it is an indispensabletool (Cremer, T. et al., In: Cold Spring Harbor Symposia on QuantitativeBiology, Volume LVIII, pp. 777-792, Cold Spring Harbor Laboratory Press,NY (1994)). Most importantly FISH offers the capacity for multiparameterdiscrimination. This allows the simultaneous visualization of severalDNA probes using either a combinatorial (Nederlof, P. M. et al.,Cytometry 10:20-27 (1989); Nederlof, P. M. et al., Cytometry 11:126-131(1990); Ried, T. et al., Proc Natl Acad Sci (U.S.A.) 89:1388-1392(1992a); Ried, T. et al., Hum Mol Genet 1:307-313 (1992b); Lengauer, C.et al., Hum Mol Genet 2:505-512 (1993); Popp, S. et al., Human Genetics92:527-532 (1993); Wiegant, J. et al., Cytogenet Cell Genet 63:73-76(1993)) or a ratio labeling (Dauwerse, J. G. et al., Hum Mol Genet1:593-598(1992); Nederlof, P. M. et al., Cytometry 13:839-845 (1992); duManoir, S. et al., Hum Genet 90:590-610 (1993)) strategy. Up to twelveDNA probes have been visualized (Dauwerse, J. G. et al., Hum Mol Genet1:593-598 (1992)). Consequently, the goal of 24 different colors haslong been sought (Ledbetter, D. H., Hum Mol Genet 5:297-299 (1992)).Twenty-four different colors are an important threshold because theywould allow the simultaneous visualization of the 22 autosomes and bothsex chromosomes. Beside improved karyotyping, the possibility ofsimultaneously hybridizing 24 different and distinguishable DNA probeswould allow the addressing of a large number of important biologicalquestions. However, the previously published multicolor systems lackedthe versatility for an extension to 24 colors and onlyproof-of-principle experiments were ever published.

[0050] The present invention results, in part, from the realization ofmultiparametric fluorescence in situ hybridization to achieve thesimultaneous visualization of 24 different genetic targets with acombinatorial labeling strategy. This strategy permits discriminationbetween many more target sequences than there are spectrallydistinguishable labels. The simplest way to implement such labeling isusing a simple “Boolean” combination, i.e., a fluor is either completelyabsent (i.e. the value of “0” will be assigned) or present in unitamount (value of 1). For a single fluor A, there is only one usefulcombination (A=1) and for two fluors A and B, there are 3 usefulcombinations (A=1/B=0; A=0/B=1; A=1/B=1). There are 7 combinations of 3fluors, 15 combinations of 4 fluors, 31 combinations of 5 fluors, 63combinations of 6 fluors, and so on (n fluorophores permitting thelabeling of 2^(n)-1 chromosomes). Thus, to uniquely identify all 24chromosome types in the human genome using chromosome painting probesets, only 5 distinguishable fluors are needed (31 total combinations).If each probe set is labeled with one or more of five spectrallydistinct fluorophores in a combinatorial fashion, simple Booleancombination can be used to identify each DNA probe by a spectralsignature dictated by its fluorophore composition (Speicher, M. R. etal, Nature Genet 12:368-375 (1996), which reference is hereinincorporated by reference).

[0051] B. Terminology of the Invention

[0052] The invention concerns a set of combinatorially labeledoligonucleotide probes, each member thereof: (i) having a predeterminedlabel distinguishable from the label of any other member of the set, and(ii) being capable of specifically hybridizing with one predeterminedautosomal or sex chromosome of a human karyotype. In the most preferredembodiment, the set will have a sufficient number of members to becapable of specifically and distinguishably hybridizing each autosomalor sex chromosome of said human karyotype to at least one member. Asused herein, the term “karyotype” denotes the compliment of chromosomesfound in a normal or aberrant cell. In a normal cells, the number ofchromosomes is 46, comprising 22 pairs of autosomal chromosomes and 2sex chromosomes (either 2 X chromosomes (if female) or an X and Ychromosome (if male)). The labels are said to be distinguishable in thatthe particular label of any one member of the set (and the identity ofthat member) differ from the particular label and identity of any othermember of the set. Since each probe member is capable of specificallyhybridizing to only one chromosome (or sub-chromosomal region) and sincethe identity of the label and probe are known in advance, the detectionof a particular label associated with an unidentified chromosomal regionmeans that the probe bearing that label has become hybridized to theunidentified chromosomal region. Since the chromosome to which thatprobe specifically hybridizes is known, the detection of adistinguishable label permits the identification of the chromosomalregion.

[0053] More specifically, the invention concerns fluors that can be usedto label oligonucleotide probes so that such probes may be used inmultiparametric fluorescence in situ hybridization. As used herein, a“fluor” or “fluorophore” is a reagent capable of emitting a detectablefluorescent signal upon excitation. Most preferably, the fluor iscoupled directly to the pyrimidine or purine ring of the nucleotides ofthe probe (Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392(1992), herein incorporated by reference; U.S. Pat. Nos. 4,687,732;4,711,955; 5,328,824; and 5,449,767, each herein incorporated byreference. Alternatively, the fluor may be indirectly coupled to thenucleotide, as for example, by conjugating the fluor to a ligand capableof binding to a modified nucleotide residue. The most preferred ligandsfor this purpose are avidin, streptavidin, biotin-binding antibodies anddigoxigenin-binding antibodies. Methods for performing such conjugationare described by Pinkel, D. et al., Proc. Nat'l. Acad. Sci. (U.S.A.)83:2934-2938 (1986), herein incorporated by reference).

[0054] The term “multiparametric fluorescence” denotes the combinatorialuse of multiple fluors to simultaneously label the same chromosome orsub-chromosomal fragment, and their detection and characterization.Chromosomes or sub-chromosomal fragments are said to be simultaneouslylabeled if they are exposed to more than a single chromosome-specificprobe under conditions sufficient to permit each chromosome-specificprobe to independently hybridize to its target chromosome. As usedherein, it is thus unnecessary for all such hybridization reactions tocommence and conclude at the same instant. The simultaneous labelingpermitted by the present invention is thus in contrast to protocols inwhich chromosomes are exposed to only a single chromosome-specific probeat a time.

[0055] The simultaneous detection and characterization permitted by thepresent invention denotes an ability to detect multiple (and mostpreferably all) of the autosomal and/or sex chromosomes in a sample,without any need to add further reagent, or probe after the detection ofthe first chromosome.

[0056] In the simplest embodiment, digital images of the chromosomes areobtained for each fluorophore employed, thereby providing a series ofgray scale fluorescence intensities associated with each fluorophore andeach chromosome. The final image is obtained by pseudocoloring theblended gray scale intensities for each chromosome.

[0057] The invention thus provides a method of simultaneouslyidentifying and distinguishing the individual autosomal and sexchromosomes of a human karyotype which comprises contacting apreparation of chromosomes, that has been previously treated to renderit in single-stranded form, with the above-described set ofcombinatorially labeled oligonucleotide probes, under conditionssufficient to permit nucleic acid hybridization to occur.

[0058] Such treatment causes at least one of each autosomal or sexchromosome of the preparation to become hybridized to at least onemember of said set of probes. For each chromosome of the preparationhybridized to a member of the set of probes one next detects andidentifies the predetermined label of that member and correlates theidentity of the label of that member with the identity of the autosomalor sex chromosome of said human karyotype with which that memberspecifically hybridizes. This process identifies the chromosomehybridized to the member. This last step is repeated until each or adesired number of autosomal and sex chromosome of the human karyotypehas been identified in the preparation.

[0059] The oligonucleotide probes used in accordance with the methods ofthe present invention are of either of two general characteristics. Inone embodiment, such probes are chromosome or sub-chromosome specific(i.e., they hybridize to DNA of a particular chromosome at lowerc_(O)t^(1/2) than with DNA of other chromosomes; c_(O)t^(1/2) being thetime required for one half of an initial concentration (c_(O)) of probeto hybridize to its complement). Alternatively, such probes are feature(e.g., telomere, centromere, etc.) specific. Such probes, being proximalto the telomere, are capable of defining and identifying translocationsthat may be so close to the chromosomal termini as to be otherwisecryptic.

[0060] Both types of probes may be used if desired. Sources of suchprobes are available from the American Type Culture Collection, andsimilar depositories.

[0061] The oligonucleotide probes used in accordance with the methods ofthe present invention are of a size sufficient to permit probepenetration and to optimize reannealing hybridization. In general,labeled DNA fragments smaller than 500 nucleotides in length, and morepreferably of approximately 150-250 nucleotides in length, probes areemployed. Probes of such length can be made by synthetic orsemi-synthetic means, or can be obtained from longer polynucleotidesusing restriction endonucleases or other techniques suitable forfragmenting DNA molecules. Alternatively, longer probes (such aspolynucleotides) may be employed.

[0062] Most preferably, the oligonucleotide probes are synthesized so asto contain biotinylated or otherwise modified nucleotide residues.Methods for accomplishing such biotinylation or modification aredescribed in U.S. Pat. Nos. 4,687,732; 4,711,955; 5,328,824; and5,449,767, each herein incorporated by reference. Biotinylatednucleotides and probes are obtainable from Enzo Biochem, BoehringerMannheim, Amersham and other companies. In brief, such biotinylated orotherwise modified nucleotides are produced by reacting a nucleoside ornucleotide with a mercuric salt under conditions sufficient to form amercurated nucleoside or nucleotide derivative. The mercurated productis then reacted in the presence of a palladium catalyst with a moiety(e.g., a biotin group) having a reactive terminal group and comprisingthree or more carbon atoms. This reaction adds the moiety to the purineor pyrimidine ring of the nucleoside or nucleotide.

[0063] In a highly preferred embodiment, such modified probes are usedin conjunction with competitor DNA in the manner described by Ward etal. (WO90/05789), herein incorporated by reference. Competitor DNA isDNA that acts to suppress hybridization signals from ubiquitous repeatedsequences present in human and other mammalian DNAs. In the case ofhuman DNA, alu or kpn fragments can be employed, as described by Ward etal. (WO90/05789). Initially, probe DNA bearing a detectable label andcompetitor DNA are combined under conditions sufficient to permithybridization to occur between molecules having complementary sequences.As used herein, two sequences are said to be able to hybridize to oneanother if they are complementary and are thus capable of forming astable anti-parallel double-stranded nucleic acid structure. Conditionsof nucleic acid hybridization suitable for forming such double strandedstructures are described by Maniatis, T., et al. (In: Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y. (1982)), by Haymes, B. D., et al. (In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985),and by Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392(1992)). For the purpose of the present invention, the sequences neednot exhibit precise complementarity, but need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure. Thus, departures from complete complementarity arepermissible, so long as such departures are not sufficient to completelypreclude hybridization and formation of a double-stranded structure.

[0064] The quantity of probe DNA combined with competitor DNA isadjusted to reflect the relative DNA content of the chromosome target.For example, as disclosed by Ward et al. (WO90/05789), chromosome 1contains approximately 5.3 times as much DNA as is present in chromosome21. Thus, a proportionally higher probe concentration would be employedwhen using chromosome 1 specific probes.

[0065] The resulting hybridization mixture is then treated (e.g., byheating) to denature the DNA present and is incubated at approximately37° C. for a time sufficient to promote partial reannealing. The samplecontaining chromosomal DNA to be identified is also heated to render itsusceptible to being hybridized to the probe. The hybridization mixtureand the sample are then combined, under conditions sufficient to permithybridization to occur. Thereafter, the detection and analysis of thehybridized product is conducted by detecting the fluorophore label ofthe probe in any of the methods described below.

[0066] In an alternative embodiment, a modification of the method ofRied T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992),herein incorporated by reference) is employed. Thus, probes are labeledeither directly (e.g., with fluorescein) or indirectly (e.g., withbiotinylated nucleotides or other types of labels), and permitted tohybridize to chromosomal DNA. After hybridization, the hybridizedcomplexes are incubated in the presence of streptavidin, that had beenconjugated to one or more fluors. The streptavidin binds to thebiotinylated probe of the hybridized complex thereby permittingdetection of the complex, as described below.

[0067] C. The Preferred Fluorophores of the Invention

[0068] By labeling with two or more fluors in combination, it ispossible to discriminate between many more objects than there areavailable fluors. The simplest way to implement such labeling is byBoolean combination, i.e., a fluor is either completely absent (0) orpresent in unit amount (1). For a single fluor A, there is only oneuseful combination (A=1). For two fluors A, B there are 3 usefulcombinations (A=1, B=0; A=0, B=1; A=1, B=1). For three fluors A, B, C,there are 7 combinations (A=1, B=0, C=0; A=0, B=1, C=0; A=0, B=0, C=1;A=1, B=1, C=0; A=1, B=0, C=1; A=0, B=1, C=1; A=1, B=1, C=1). There are15 combinations of 4 fluors, 31 combinations of 5 fluors, 63combinations of 6 fluors, and so on.

[0069] To uniquely code all 24 chromosome types in the human genome, 5distinguishable combinatorial fluors are needed. With a 5-fluor set, 15chromosomes can be distinguished using combinations of 4 of the 5fluors. The labeling of the remaining 9 chromosomes requires all fivefluors to be used combinatorially. Seven of the available 5-fluorcombinations are not required. Thus, there is a certain amount oflatitude available to avoid any 5-fluor combination that might proveparticularly hard to resolve. In particular, quaternary or quinternarycombinations may be avoided.

[0070] One aspect of the present invention concerns the identificationof a set of seven fluors that are be well resolvable by theexcitation-emission contrast (EEC) method.

[0071] As indicated above, multi-fluor combinatorial labeling depends ingeneral on acquiring and analyzing the spectral signature of each objecti.e., obtaining the relative weighting coefficients of the componentfluors. Because full spectroscopic analysis, of mixed fluor spectra(e.g., by interferometry) is not yet sufficiently developed, the methodchosen was conventional bandwidth-restricted widefield imaging usingepi-fluorescence triplets, viz. excitation filter, dichroic reflectorand emission bandpass filter. The limited spectral bandwidth availablefor imaging (roughly 380-750 nm), and the extensive overlap between thespectra of organic fluors, makes separating multiple fluorsspectroscopically during the imaging step a significant technicalchallenge.

[0072] To make software segmentation of the source images asstraightforward as practicably possible, a target figure of <10%crosstalk between any given fluor and the two adjacent channels was set.Computer modeling indicated that for DAPI plus the five combinatorialfluors FITC, Cy3, Cy3.5, Cy5, Cy5.5, this level of contrast cannot beattained using either excitation selection or emission selection alone,no matter how narrow the filter bandwidths. Thus, both excitationselection and emission selection must be invoked simultaneously. This isreferred to as excitation-emission contrast (EEC).

[0073] Contrast ratio plots were first computed for each of the fluorsvs. its two neighbors. These plots indicate regions where pairwisecontrast is high enough to be useful. A constraint on the practicallyattainable contrast is that regions of high contrast generally lie fardown the flanks of at least one of the spectra i.e., where excitationand/or emission are strongly sub-optimum. Further, to attain therequired degree of selectivity it is necessary to use filters ofbandwidths in the range 5-15 nm (cf. approx. 50 nm for ‘standard’ filtersets). Together, these impose a severe sensitivity penalty. The goal of10% maximum crosstalk represents an acceptable, practical compromisebetween sensitivity and selectivity.

[0074] A fundamental asymmetry exists between excitation contrast andemission contrast. For low-noise detectors such as the cooled CCD,restricting the excitation bandwidth has little effect on attainableimage S/N ratio; the only penalty is the need for longer exposure times.Restricting the emission bandwidth is very undesirable, however, sinceevery fluorescence photon blocked by the filter represents irreversiblephotochemical bleaching of the fluor. For this reason, the highestpracticable contrast was invoked on the excitation side. A suitableemission filter was then found to give the necessary EEC ratios. Filterselection is additionally constrained by the fact that each channel mustadequately reject both adjacent channels simultaneously: improving onemay significantly degrade the other. Good contrast was attainable inpractice for all fluors except the Cy5.5/Cy5 pair, which is marginal.For this reason, Cy7 was later substituted for Cy5.5. Otherconsiderations relating to choice of filters include:

[0075] 1. Commercial narrow-band interference filters may have a largeamount of wedging i.e., non-parallelism between the top and bottomfaces. This results in large image shifts (up to several micronsequivalent). The shift is a vector characteristic of each filter and itsorientation in the epicube. Thus, automatic compensation for imagedisplacement is a necessary part of the processing software.

[0076] 2. Manufacturing variations of a few nm in peak wavelength andFWHM specifications can have significant effects on the EEC ratios.Filter errors to long wavelength may be fine-tuned by tilting, but thisoption is severely curtailed in the case of emission filters because ofincreased image aberrations and worsened pixel shifts. There is noequivalent way to compensate short-wavelength errors.

[0077] 3. the need to prevent infra-red light emitted by the arc lampfrom reaching the detector. Silicon CCD's are extremely sensitive inthis region. Filter sets for the blue and midvisible fluors were foundnot to need additional IR blocking, but loss of image contrast due tospurious IR was found to be a serious problem for the red—far red—nearIR fluors. Heat filters routinely used in microscopy (e.g., Schott BG-38glass) are completely inadequate to alleviate this problem. Thus,extensive additional blocking was required. However, availablecommercial interference filters for infra-red blocking filters alsotransmit poorly in the near UV, and thus cannot be inserted in theexcitation path. Instead, it was found necessary to put the IR blockingfilters into the emission path. To minimize loss of image quality byinsertion of these filters in the image path, they are placed inside theCCD camera, immediately in front of the window. In practice, twointerchangeable filters were chosen, one for use with Cy5, Cy5.5 (Oriel#58893; 740 nm cutoff) and one for use with Cy7 (Oriel 58895; 790 nmcutoff).

[0078] The first member of the set of fluors is the counterstain DAPI,which gives a weak G-like banding pattern. Five of the remaining sixfluors may be used combinatorially to paint the entire human chromosomeset. All are available as avidin conjugates (for secondary detection ofbiotinylated probe libraries) or directly linked to dUTP (for directlabeling).

[0079] Thus, a set of six fluors and corresponding optical filtersspaced across the spectral interval 350-750 nm was identified thatachieve a high discrimination between all possible fluor pairs. Thesefluors comprise the preferred fluors of the present invention and are:4′-6-diamidino 2-phenyl indole (DAPI), fluorescein (FITC), and the newgeneration cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Of these Cy3,Cy3.5, Cy5 and Cy7 are particularly preferred, The absorption andemission maxima for the respective fluors are: DAPI (Absorption maximum:350 nm; Emission maximum: 456 nm), FITC (Absorption maximum: 490 nm;Emission maximum: 520 nm), Cy3 (Absorption maximum: 554 nm; Emissionmaximum: 568 nm), Cy3.5 (Absorption maximum: 581 nm; Emission maximum:588 nm), Cy5 (Absorption maximum: 652 nm; Emission maximum: 672 nm), Cy7(Absorption maximum: 755 nm; Emission maximum: 778 nm). Completeproperties of selected fluorescent labeling reagents are provided byWaggoner, A. (Methods in Enzymology 246:362-373 (1995) hereinincorporated by reference). In light of the above, it is readilyapparent that other fluorophores and filter combinations having adequatespectral resolution can alternatively be employed in accordance with themethods of the present invention.

[0080] D. Methods for the Detection of Fluorescent In situ Hybridization

[0081] 1. The Theory of Fluorescence Detection

[0082] Of the various methods for contrast generation in site-specificlabeling, fluorescence is arguably the most powerful, because of itshigh absolute sensitivity and multiparameter discrimination capability.Modem electronic cameras used in combination with high numericalaperture microscope objectives and state of the art optical filters arecapable of imaging structures labeled with as little as a 10-100 fluormolecules per pixel. Thus, fluor-tagged single-copy DNA sequences assmall as a few hundred bases in size are detectable under favorableconditions. The availability of families of spectrally distinguishablefluors makes simultaneous imaging of several different targets in thesame specimen possible, either directly or through combinatorial oranalog multiplex methods. In principle, multi-fluor discrimination maybe based on differential excitation of the fluors, differentialemission, fluorescence lifetime differences, or on more complex butstill analyzable observables such as fluorescence anisotropy. Thisdiscussion assumes an epi-imaging geometry. Table 1 describes thesymbols and operators relevant to the theoretical considerations offluorescence. TABLE 1 Units Definition Instrument Parameter ψ_(s)(λ)photon s⁻¹ nm⁻¹ spectral distribution of source, assumed to be anisotropic radiator over 4π steradians φ_(s) dimensionless collection ofefficiency of condenser optics ψ1(λ) photon s⁻¹ m⁻² nm⁻¹ spectraldistribution of photons in collimated beam of excitation light impingingon excitation filter F1 f1(λ) dimensionless transmittance function ofexcitation filter F1 ψ2(λ) photon s⁻¹ cm⁻² nm⁻¹ spectral distribution ofphotons in collimated beam of excitation light emerging from F1 andimpinging on dichroic beamsplitter DB1 R(λ) dimensionless reflectancefunction of dichroic beamsplitter DB1 T(λ) dimensionless transmittancefunction of dichroic beamsplitter DB1 ψ3(λ) photon s⁻¹ cm⁻² nm⁻¹spectral distribution of photons in collimated beam of fluorescenceemerging from DB1 and impinging on emission filter F2 f2(λ)dimensionless transmittance function of emission filter F2 ψ4(λ) photons⁻¹ cm⁻² nm⁻¹ spectral distribution of photons in collimated beam ofexcitation light emerging from F2 and entering the entrance pupil of theobjective lens μ dimensionless linear magnification factor of objectivelens ψ5(λ) photon s⁻¹ cm⁻² nm⁻¹ spectral distribution of photons infocused beam of excitation light traversing the specimen plane, φ_(d)(λ)dimensionless quantum efficiency of detector Ω pixel.μ⁻¹ magnificationfactor of final image at detector Fluor Parameter ε(λ) M⁻¹ cm⁻¹ molardecadic extinction coefficient of fluor F_(a). τ s decay lifetime ofexcited state of fluor F_(a). σ_(Fa)(λ) cm⁻¹ photon absorptioncross-section of fluor F_(a). φFa dimensionless fluorescence quantumefficiency of fluor F_(a). ƒ _(a)(λ) dimensionless normalized spectraldistribution of fluorescence from fluor F_(a).

[0083] Whether a given excitation rate at pixel location p will give anacceptable fluorescence signal/noise ratio (defined as S/N=[signalmean]/[variance due to all noise sources]) in a given integration perioddepends on the number of fluor molecules within p, their quantum yieldand photochemical stability, and the quantum efficiency and noiseperformance of the detector.

[0084] In the limit of weak excitation (ψ^((λ)).σ_(F) ^((λ))<<τ⁻¹), therate of excitation of the N molecules of fluor F within pixel p inobject space is: R(p) = N∫_(i  ψ)5(λ, p) ⋅ σ_(F)(λ) ⋅ λ  s⁻¹

[0085] ψ^(5(λ)) is the spectral distribution of exciting light passingthrough the focal plane of the microscope (photon nm⁻¹.cm⁻².s⁻¹). It isapproximately given by ψ_(s) ⁽⁸⁰⁾.φ_(s).f1(λ).R(λ).α².μ, whereα=diameter of objective lens entry pupil/diameter of collimated beamfrom condenser. The integral is taken over the bandwidth (i) for whichthe fluor has non-zero absorption cross section π_(F) ^((λ)), which isrelated to the molar decadic extinction coefficient ε_(F)(λ) by theexpression:

σ_(F) ^((λ))=3.825×10⁻²¹ε_(F)(λ)cm ².

[0086] In practice, ∫_(i)ψ^(5(λ,p)).dλ can be measured with a bolometricdetector such as a calibrated micro-thermopile placed at or near thefocal plane of the objective lens.

[0087] For a perfectly noiseless detector and a non-bleachable fluor,the S/N of each pixel increases indefinitely as (photonsdetected)^(1/2). How rapidly S/N increases depends on excitationstrength, but the relationship between S/N and dose does not. The effectof non-zero bleaching constant is to change the t^(1/2) function to anasymptotic function, the form of which depends on the bleachingmechanism. However, because the bleaching rate and the signal strengthare linearly related, the asymptote once again does not depend at all onexcitation rate, although the speed of approach to the asymptote does.If finite camera noise is added to photobleaching, it is found that theS/N climbs to a maximum value, then falls as the fluor is exhausted. Nowboth the kinetics and the peak S/N depend of excitation rate, in generalthe faster the excitation the higher the maximum attainable S/N.However, for contemporary cooled CCD cameras the dark noise is so lowthat it can be virtually ignored on the timescale of bleaching(typically a few minutes); a more important factor in determiningultimate S/N is the stray light background (esp. from nonspecificluminescences and leakage of excitation light).

[0088] Note that although the microscope objective compresses theexcitation beam, in a nonconfocal microscope it does not focus it to apoint and so has no bearing on the fluorescence image resolving power(incoherent emitter).

[0089] The commonest excitation source for fluorescence microscopy isthe high pressure short mercury arc, whose spectrum consists ofpressure-broadened lines from the UV to the middle region of the visiblespectrum (principal wavelengths are 334.1 nm, 365.6 nm, 404.7 mm, 435.8nm, 546.1 nm. 577.9 nm), superimposed on a weaker thermal continuum.Many fluorophores have excitation spectra that overlap one or other ofthe mercury lines to an acceptable extent. Others (of which the bestknown is FITC) do not, but may be adequately excited by the continuum ifa wide enough excitation bandwidth is employed. Another common source isthe high pressure xenon short arc, which produces an almost uniformcontinuum from ca. 300 nm: to beyond 900 nm. However, the power nm⁻¹, isalmost everywhere less than the mercury continuum for the same arcwattage. If high intensity light with no structure is required (e.g.,for fluorescence ratio imaging) a high CW power or pulsed quartz halogenlamp outperforms the xenon beyond about 450 nm. Certain fluors are wellmatched to laser excitation (e.g., Ar+@488 nm for FITC, He—Ne @632.8 nmfor Cy5, semiconductor diode—pumped YAG @680 nm for Cy5.5).

[0090] In single-fluor imaging, use of the available spectral bandwidthis rarely stringent. The excitation filter Fl and dichroic beamsplitterDBl can usually be chosen to give adequate overlap between the sourcespectrum and the fluor excitation spectrum. If an arc line is available,the Fl bandpass need be no wider than the line. If not, and part of thethermal continuum must be used, the wider the Fl bandpass the greaterwill be the available excitation flux. However, with low noiseintegrating detectors the goal of high excitation efficiency isgenerally secondary to the need to exclude excitation light from theemission path. This limits how close the excitation and emissionbandpasses can be placed to one another, and hence constrains theexcitation bandwidth. For fluors with small Stokes' shifts, high qualityfilters with very steep skirts are required. The excitation filter mustbe rigorously ‘blocked’ on the long wavelength side, and have nopinholes, scratches, or light leaks around the edge.

[0091] Dichroic beamsplitters are currently much less ‘evolved’ thanbandpass interference filters, meaning that the slopes of their transmit<-> reflect transitions are far less than the skirt slopes of premiumnotch filters, and there may be large spectral intervals where theyoscillate between intermediate states of partial reflectance andtransmittance. The main purpose of a dichroic beamsplitter is to improvethe combined efficiency of excitation and emission, rather than todefine the wavelength response of the instrument.

[0092] The resolvability of overlapping fluors in imaging microscopy maydepend critically on the degree of excitation contrast that can beachieved (see C, below). The variation with wavelength of the ratio ofthe extinction coefficient of two fluors is the excitation contrastspectrum. It can readily be calculated from the digitized absorptionspectra. Depending on the overlap of the absorptions, their ratiospectrum may either show a distinct peak or may grow indefinitely large.In either case, it is usually possible to choose an excitationwavelength that favors one fluor over another to a useful extent (from afactor of 3-4 fold up to a hundredfold or more). Some difficulties inobtaining high contrast multi-fluor images include:

[0093] a. The excitation wavelengths required for high contrast imagingare often far from the absorbance peaks. Thus, there may be a highdegree of intensity trade-off to obtain high signal contrast vs. otherfluors.

[0094] b. From the above, standard filter sets cannot be used.

[0095] c. Arc source spectral lines that are useful for exciting singlefluors may not give high contrast discrimination against adjacentfluors.

[0096] d. When using a broadband source or the continuum spectrum of anarc source, the need for narrow excitation bandwidth may reduce theexcitation flux to problematically low levels. It is sometimes possibleto relax the constraints on excitation wavelength for the sake of moreefficient excitation.

[0097] Collecting the fluorescence of F and imaging it onto the detectorwith high efficiency is the principal design goal of the emissionoptics. Operationally, it is even more important to have an efficientemission path than an efficient excitation path. The reason is thatinefficient excitation causes inefficient photobleaching; the onlypenalty is a long image integration time (assuming a low-noisedetector). On the other hand, any fluorescence lost on its way to thedetector represents photobleaching without concomitant increase in theinformation content of the image.

[0098] The detector pixel p accumulates signal (detected photons) at arate:F(p) = G ⋅ R(p) ⋅ φ_(F) ⋅ ∫_(ii)T(λ) ⋅ f2(λ) ⋅ φ_(d)(λ) ⋅ λ ⋅ s⁻¹

[0099] The integral is over the bandwidth (ii) for which the detectorquantum efficiency φ_(d)(λ)>0. G represents the efficiency with whichthe optics gather the fluorescence and transmit it to the detector; itmay be assumed to be wavelength independent to first order. Theprincipal factor in G is the numerical aperture (NA) of the objectivelens, which determines the fraction of the isotropically radiatedfluorescence collected by the imaging system (§=1/π. sin⁻¹(θ)=1/π.sin⁻¹(NA/n), where θ is the half-angle subtended by the objective lensfrom its focal point. For an oil immersion lens with NA=1.3; n=1.515;§=0.328). NA additionally determines the spatial resolving power of themicroscope, because it scales the dimensions of the Frauenhoferdiffraction pattern produced in the image plane by a point source in thespecimen plane. Several ‘rules’ are in use for specifying the resolvingpower of a lens, depending on how much overlap of the Airy discs of twoadjacent objects is deemed to constitute the threshold of resolution.The commonly used Rayleigh criterion is r=1.22λ/2NA (e.g., for the aboveNA=1.3 lens working at 500 nm, r=0.24 μ).

[0100] For a noise-free detector, image ‘noise’ at p is determined bythe statistical variance in the number S(p) of fluorescent photonsdetected in time interval Δt. The only detector characteristic that hasany bearing on this is its quantum efficiency Φd(λ).

[0101] In the absence of photobleaching (probability of destruction perhit Π_(b)=0), S(p)=F(p).Δt, with variance S(P)^(1/2), i.e.,S/N=S(p)^(1/2). Image quality will therefore increase indefinitely withΔt, though at an ever decreasing rate.

[0102] In the presence of photobleaching (Π_(b)≠0), S(p) is an integralof the form: S(p) = F(p)∫_(Δ  t)f(t) ⋅ t

[0103] where f(t) is the photobleaching decay function. S/N rises alonga more or less complex path to an asymptotic value that corresponds tototal exhaustion of the fluor. For the case of a unimolecularphotobleaching process this would be an exponential function. i.e.,N(p,t)=N₀(1−exp−[k_(b)/Σk].t) where k_(b)=Π_(b) ⁻¹ is the photobleachingconstant and Σk represents all other processes by which the excitedstate of F is deactivated. Note that the normalized asymptote in thisfirst-order system depends only on k_(b)/Σk, and is independent of thestrength of the excitation. Thus, the extent of bleaching isexponentially related to the accumulated excitation dose, but isindependent of the path. In reality, however, bleaching of fluors insolution may be mechanistically and kinetically much more complex. Acommon mechanism involves ring opening following peroxidation of thefluor excited state, e.g., by ¹O₂ or O₂ ²⁻. This type of bleaching maybe considerably slowed by rigorous deoxygenation or by the use of oxygenradical scavengers (i.e., antifade agents) such as tertiary amines(p-phenylene diamine or DABCO). Nevertheless, other (as yet poorlycharacterized) irreversible processes are not excluded, includingreactions with impurities.

[0104] A nonideal detector contributes noise of many kinds, detailedanalysis of which may be intractable. The simplest noise component isfluctuation in the so-called ‘dark current,’ i.e., the flow of thermallyexcited carriers within the detector. If this noise is assumed to berandom, it adds to the photon shot noise in RMS fashion. Thus, if themean photogenerated signal is F s⁻¹ and the mean dark count is D s⁻¹,the S/N after time Δt is F.(Δt)^(1/2)/(F+D)^(1/2); S/N still increasesas (Δt)^(1/2), but more slowly than for a noiseless detector. Whenphotobleaching is present, however, the situation is entirely different.In this case, S/N rises initially as (Δt)^(1/2), but at some pointreaches a shallow maximum and then begins to fall again, as thefluorescent signal declines but the thermal noise power remainsconstant. In this case, it is in principle desirable to continuouslymonitor the S/N of the image, and terminate the exposure when the peakis reached. Most commercial digital imaging systems make no provisionfor this. Fortunately, state of the art cooled CCD cameras have solittle dark noise per pixel (typically <0.01 electron s⁻¹ in invertedclock mode) that S/N would not begin to fall until almost completeexhaustion of the fluor. In practice, autofluorescences and stray lightdominate system performance long before the noise threshold of the CCDis reached.

[0105] The principal design goals for a single-fluor imaging system are:

[0106] 1. To achieve an adequate rate of excitation of the fluor (F).

[0107] 2. To collect the fluorescence of F and image it onto thedetector with high efficiency and with the necessary spatial resolution.

[0108] 3. To prevent reflected and/or scattered excitation light fromreaching the detector.

[0109] Design of the emission channel for a single fluor isstraightforward. The dichroic beamsplitter transition wavelength isspecified at for example, 20 mn to the red of the excitation passband.This ensures a high level of rejection of exciting light reflectedand/or scattered from the specimen and/or microscope optics. Theemission filter cut-on is usually considerably steeper than the dichroicedge, and so can be placed practically coincident with it. The mostefficient emission filter is a long-pass element. The preferred filterof this type is Schott glass, which transmits upwards of 90% of allfluorescence to the red of its cut-on, while rejecting other light(especially any excitation light that gets through the dichroicbeamsplitter) to very high order—typically >10⁵. However, it is usuallyinadvisable to leave the emission channel ‘wide open’ into the nearinfrared, especially with silicon detectors which have high sensitivitythere.

[0110] The multiparametric imaging of the present invention not onlyincreases the throughput of information about the system underobservation and makes more efficient use of the biological material, butalso can reveal spatial and temporal correlations that might otherwisebe difficult to establish reliably. When a large number of differentobjects must be visualized, two or more labels can be usedcombinatorially, which permits discrimination between many more objecttypes than there are spectrally distinguishable labels. Some examples ofmulti-fluor imaging are:

[0111] a. The co-distribution of proteins in structures such asmicrotubule networks may readily be visualized using immunolabels linkedto different fluors.

[0112] b. Multiple genes may be simultaneously mapped by fluorescence insitu hybridization (FISH) to a single metaphase chromosome spread. Suchsignals cannot usually be discriminated reliably on the basis ofintensity alone, and are usually morphologically identical(diffraction-limited points). However, they are readily discriminated bydiscrete or combinatorial multi-fluor labeling.

[0113] c. Identification of small chromosomal translocations is mostreadily done by painting with chromosome specific DNA probe librarieslinked to separable fluors, used either singly or combinatorially.

[0114] d. Analysis of mixed populations of morphologically identicalbacteria can also be achieved using species-specific DNA or ribosomalRNA probes coupled to separable fluors.

[0115] The primary design goal of a multi-fluor imager (in addition tothose for of a single fluor imager) is to spectrally resolve thefluorescence at any pixel location into components corresponding to eachfluor. Methods for spectrally resolving complex signals in fluorescentmicroscope images are outlined below.

[0116] There are several ways to resolve spectrally complex signals,i.e., to determine which fluors contribute to the fluorescence at agiven pixel location. The most general method in principle is tospectrally disperse the 2D image along a third axis, orthogonal to thex,y axes. This amounts to imaging through an optical system with a verylarge amount of chromatic aberration, such that at each position on thez axis there is an image x,y that contains only a small spectralbandwidth λ+Δλ. An area detector with very small depth of field (such asa spatially filtered confocal imager) could then be moved incrementallyalong the z axis to obtain a family of images, each containing its ownsmall spectral interval. A trace through the images at given x,y wouldconstitute an emission spectrum for that pixel. Unfortunately,implementing such a scheme is technically very difficult.

[0117] If the spectrum of only a small number of objects within theimage is required (such as individual stars in a telescope image), asolution is to extract the light corresponding to each object with aprobe (e.g., a fiber optic) and disperse it with an imaging spectrographonto a 1-dimensional array detector. To be useful in microscopy, such adevice would have to be arbitrarily positionable in the field, and havean adjustable acceptance area.

[0118] The most reasonable method for full spectral analysis inmicroscopy is to image through a variable narrow band filter. An imageis recorded at each wavelength; intensity values at a given pixellocation through the series represent a weighted emission spectrum thatcan be fit to a linear combination of the known spectra of the componentfluors. The coefficients are products of the relative molar amounts ofthe fluors with their extinction coefficients at the exciting wavelengthand their fluorescence quantum yields. If the last two are known, thefirst is obtainable from the fit. In general, it is necessary to takeseveral such image sets, at several excitation wavelengths, to get aunique fit. With enough iterations, this process generates a 3D surfaceof intensity values as a function of both excitation and emissionwavelength. This comprises a complete spectral signature of the pixel,giving a very highly constrained solution for the relative amounts ofits component fluors. The mole ratios could be mapped back onto the x,ycoordinates of the image, with appropriate pseudocolor coding, to give a‘composition map’. This general (and quantitative) method has a numberof technical difficulties, although none of them is insurmountable. Thefirst is that a large number of images are required to evaluate a singlemicroscope field. Imaging time is long, and extensive differentialphotobleaching of the fluors would make it impossible to achieve a selfconsistent “fit” to the spectral data. Instrument stability is also anissue, particularly with arc sources, the output spectra of which changethroughout their life. Finally, the amount of computation required togenerate a composition map would realistically limit the analysis tosmall image regions only.

[0119] For most applications, there is no need of a full-blown spectralanalysis capability because the fluors to be analyzed are known ahead oftime. Thus, it is only necessary to be able to identify which fluor iswhich, and, for multiplex imaging, to ascertain the relative amount ofeach present. A preferred approach to this category of problems involvesthe use filter sets that achieve a high degree of selective excitationand visualization of each component fluor. The ideal system of this typewould perfectly isolate each fluorophore, i.e., “one image—one fluor”and all off-diagonal elements in the matrix of intensity coefficientswould be zero. However, the facts that the spectra of typicalfluorophores are 10-30% of the total available bandwidth, and emissionfilters must have significant bandwidths to pass usable amounts of lightplace severe limitations on the attainable degree of spectral isolation.Nevertheless, it is not difficult to achieve useful levels of contrastbetween suitably chosen fluors, such that residual crosstalk can beremoved numerically.

[0120] The excitation-emission contrast (EEC) approach is in principleapplicable to analysis of images involving multiple fluors withfine-grained distributions of mole fraction (e.g., fluorescence ratioimaging), subject to image S/N and the limitations of differentialbleaching rates and source instability. However, it is particularlysimple for the limiting case of combinatorial labeling, in which fluorsare multiplexed in a strictly Boolean fashion (present=1, or absent=0).In this case, it is necessary only to be able to reliably discriminatebetween a 1 and a 0, for which a 10:1 intensity ratio between any fluorand its neighbors would be sufficient. However, for many useful fluorsthis is not achievable on the basis of excitation contrast or emissioncontrast alone. Simultaneous selection on the basis of both excitationand emission are required.

[0121] In some circumstances it is required to excite and image severalfluors simultaneously, e.g., for direct viewing and/or color videorecording. Excitation and emission optics that have no wavelengthselectivity cannot be used, because the excitation light scattered intothe detector would overwhelm the fluorescence by several orders ofmagnitude. One solution is to use multiple-bandpass filters designed forthe specific set of fluors to be used. The excitation filter definesnarrow passbands that overlap the fluor excitation spectra. The emissionfilter defines similar passbands that interdigitate between theexcitation bands and overlap the fluor emission spectra (the reddestfluor could use a long-pass filter). The dichroic beamsplitteralternates between reflect (overlapping the excitation passbands) andtransmit (overlapping the emission passbands).

[0122] Use of a multi-pass filter set also has an advantage even whenmulticolor visualization is not required, viz. the absence of geometricdisplacement (pixel shifts) between signals passing through the severalemission passbands. However, when used with a greyscale imaging system,it is necessary to make either the excitation filter or the emissionfilter switchable, in order to determine which greyscale signalcorresponds to which fluor. The preferred choice is to switch theexcitation filter, because this causes no image displacement.

[0123] A second theoretical solution would be to excite at a wavelengthwhere all the fluors absorb. This is often possible because many fluorsare excitable to states higher than S 1 using photons in the middle UV,but because of internal relaxation processes give ‘normal’ fluorescence.For example, many laser dyes can be excited at the nitrogen laserwavelength, 337 nm, far to the blue of their visible absorbances. Itwould be straightforward to block such exciting light from the emissionpath, using a long-pass filter (e.g., 380 nm), while allowing allfluorescences to simultaneously reach the detector. Drawbacks to the useof UV excitation include increased rates of photochemical decompositionof the fluor, and the expense of suitable UV optics. Thus, the methodhas not found widespread use.

[0124] The multi-bandpass method has the limitation that construction ofmultiple bandpass elements giving adequate contrast between more than 3fluors is extremely difficult. A generally more powerful approach is toconstruct optimized filter sets for each fluor, and switch them asneeded. In the case of a single fluor, the primary goal on theexcitation side is high excitation flux, to give a bright image. Whenimaging multiple fluors, however, this becomes secondary to the goal ofachieving high contrast between fluors. In the weak overlap limit,fluors may be imaged by sequentially switching filters that are designedusing the same criteria as single-fluor sets (except that long-passemission filters are proscribed for all but the longest-wavelengthfluor). Crosstalk of a few percent is usually allowable, and can becompensated numerically if necessary. More generally, though, generationof adequate contrast between fluors requires both strongly selectiveexcitation and emission. The optimum wavelengths can be found byratioing the spectra of adjacent fluors. These wavelengths may be farfrom the excitation maxima, implying a significant tradeoff betweensignal brightness and contrast. Standard filter sets cannot be used. Theneed for precisely placed excitation makes it less likely that a strongexcitation line will be available, and also reduces the flux availablefrom a continuum source. It may be necessary to pulse the broadbandsource (arc or filament lamp) to transiently very high output levels, orto supplement it with laser sources.

[0125] The principal technical problem with serial imaging is imagedisplacement when filters are changed i.e., the coordinate systems ofthe individual members of an image series are not in preciseregistration. This problem arises from nonidealities in the emissionchannel optics. Two displacement components can be identified:

[0126] i. a reproducible offset that is unique to each filter set. Thiscomponent is a fixed vector, and arises mainly from imperfectparallelism (i.e., wedging) between the top and bottom faces of theemission bandpass filter. There is also a small component due to wedgingin the dichroic beamsplitter, but since this element is very thin theeffect is minor. Since the wedging vector is a constant for each filterset, it can be automatically removed in the computer. The size of theoffset can also be reduced to very small values (<0.1 μ) by selectingemission filters for a high degree of parallelism e.g., by measurementin a laser autocollimator.

[0127] ii. a random component due to vibration and hysteresis in thefilter switching mechanism. The magnitude of the noise depends on thefilter switching mechanism. The worst are manual push-pull sliders,particularly when the operator actuates them using uncompensated forces.The best are motorized filter cassettes, in which all mechanical torquesact against the microscope body.

[0128] Note that both the constant and random components of the imageoffset noise are minimized by using an objective lens of the highestpossible magnification, and the minimum amount of magnification in thecamera projection optics. Image displacements encountered in opticalmicroscopy are linear vectors only; there is no evidence for rotation orsignificant changes in scale (magnification).

[0129] Design considerations for high contrast imaging of multipleoverlapping fluors are similar to those already outlined for theexcitation channel. By calculating the ratio of each fluor's emissionspectrum to that of its neighbors, it is possible to identify spectralregions of high contrast that can be defined with narrowband filters.High contrast is often achievable only at the cost of throwing awaysignificant amounts of the fluorescence (90% or more). Inefficiency inthe fluorescence channel is much more damaging to image S/N thanexcitation Inefficiency. Thus, where possible, selective excitation isthe preferred method of achieving contrast between multiple fluors. Indifficult cases, however, both excitation and emission contrast arerequired.

[0130] 2. Optical Filter

[0131] As indicated, in a preferred embodiment of the invention, thedetection of fluor is accomplished using optical filters, in amodification of the method of Ried, T. et al. (Proc. Natl. Acad. Sci.(U.S.A.) 89:1388-1392 (1992), herein incorporated by reference).

[0132] Imaging DAPI

[0133] 4′,6-diamidino-2-phenylindole (DAPI) is a commonly used DNAcounterstain that intercalates preferentially into AT-rich regions ofchromosomes and so gives rise to a weak G-type banding pattern. It is avery bright fluor (ε=3.3×10⁴ M⁻¹ cm⁻¹ at 347 nm, with approx. 20-foldenhancement of fluorescence quantum yield when intercalated into theminor groove of double-stranded DNA), and is relatively resistant tophotobleaching. The following points are relevant to imaging DAPI withhigh contrast against its neighbors:

[0134] a. DAPI has very broad excitation and emission spectra, and avery large Stoke's shift. Thus, although the DAPI excitation maximum(347 nm) is to the blue of Cascade Blue (CB), the fluorescence of DAPIpeaks to the red of Cascade Blue, and actually overlaps quite stronglywith FITC.

[0135] b. The usual excitation wavelength for DAPI (Hg 366 nm line)cannot be used in this application, because it is almost isosbestic forDAPI and CB and thus gives no excitation contrast.

[0136] c. The peak of the DAPI/CB excitation contrast spectrum is at 320nm, which is too far into the UV to pass through the microscope optics.An acceptable compromise is to excite with the Hg 334.1 nm line, whichthe microscope transmits with about 30% efficiency. The Ealing 35-2989interference filter has an appropriate bandpass. The excitation contrastratio for DAPI/CB at 334.1 nm has an absolute value of 4.0.

[0137] d. To further increase the DAPI/CB selectivity, emission contrastmust be used in addition to excitation contrast. Note that DAPI emitsthe bulk of its fluorescence to the red of CB. The wavelength of maximumemission contrast for DAPI vs. both CB and FITC is 490 nm. A suitableimaging-grade filter is the Omega 485DF22. There are no mercury lineswithin its bandpass, so good DAPI images with low flare are expected.The calculated emission contrast between DAPI and both Cascade Blue andFITC is 6.8. Hence, the overall contrast achievable for DAPI vs. CascadeBlue is approximately 27-fold. The overall contrast of DAPI vs. FITC isvery much higher than this, because at the DAPI excitation wavelengththe excitation of FITC is close to zero.

[0138] e. The Omega 450DRLP02 dichroic beamsplitter is very well matchedto the proposed excitation and emission filters.

[0139] Imaging Cascade Blue

[0140] Cascade Blue (CB) has a broad, two-peak excitation spectrum thatoverlaps DAPI extensively, though not its neighbor on the red side(FITC). The Stokes shift for CB is very small, i.e., there is veryextensive overlap between its excitation and emission spectra. Thesefactors combine to make imaging CB in the presence of DAPIproblematical. To summarize:

[0141] a. The peak of the CB/DAPI excitation contrast spectrum is at396-404 nm. However, because of the very small Stokes' shift of CB thisis very close to the emission contrast peak (408 nm). Since it is moreimportant for image S/N to collect the emission with high efficiencythan to excite with high efficiency, the 400-410 nm region is reservedfor the CB fluorescence. The best compromise for CB excitation is theOmega 380HT15, which overlaps with the Hg 366 nm line enough to providegood excitation strength.

[0142] b. To achieve any emission contrast at all vs. DAPI, the emissionfilter must be narrow and very carefully placed. A suitable filter isthe Omega 405DF10, but the theoretical maximum contrast is only 2.5.Thus, imaging CB while excluding DAPI is expected to be marginal.

[0143] c. Cascade Blue exhibits high excitation contrast vs. FITC(contrast ratio with the Omega 380HT15 filter=6).

[0144] d. The emission contrast ratio for CB/FITC goes to very largevalues below 490 nm, and is essentially infinite at the position of the405DF10 filter.

[0145] The above considerations indicate that it will be possible to useeither DAPI or Cascade Blue, but not both together unless the DAPIcounterstain is weak. Cascade Blue is well separable from FITC and theother five combinatorial fluors considered herein.

[0146] Imaging FITC

[0147] The following points are relevant to high-contrast imaging ofFITC:

[0148] a. The excitation spectrum of FITC has insignificant overlap withthat of Cascade Blue (contrast parameter Rb for FITC/CB becomesextremely large beyond 420 nm). This makes it possible to excite FITC onthe high frequency shoulder of its absorbance, so avoiding appreciableexcitation of Cy3.

[0149] b. There is no mercury line that overlaps well with the FITCexcitation spectrum. Thus, it is necessary to use the continuum toexcite this fluor, as with single-fluor imaging. The gap between FITCand CB makes it possible to use a very wide excitation filter; the Omega455DF70 is well suited to this function.

[0150] c. The Omega 455DF70 bandpass also corresponds fortuitously tothe maximum in the excitation contrast ratio for FITC/Cy3 (460 nm;absolute extinction ratio R_(a)=8.8).

[0151] d. The emission spectrum of FITC overlaps that of Cy3 (on the redside) and both DAPI and CB (on the blue side) to an appreciable extent.However, because the conditions defined for FITC excitation do notexcite DAPI or Cascade Blue at all, they are not relevant. The FITC/Cy3emission contrast ratio goes to very high values below 540 nm. Thus, theOmega 530DF30 filter gives very high emission contrast, which compoundsthe high excitation contrast ratio for FITC vs. Cy3 (8.8) given by the455DF70. It therefore appears possible to image FITC very cleanly.

[0152] Imaging Cy3

[0153] The following points are relevant to high-contrast imaging ofCy3:

[0154] a. The absorbance peak of Cy3 is at 551 nm, at which wavelengththe excitation of FITC is essentially zero (contrast parameter Rb jumpsto extremely high values to the red of 525 nm).

[0155] b. The Cy3 extinction peak overlaps strongly with the Hg 546.1 nmline.

[0156] c. The excitation contrast ratio for Cy3/Cy3.5 is everywheresmall, and varies weakly with wavelength. At 551 nm, the absolute valueof the excitation contrast for Cy3 vs. Cy3.5 is less than 2, and it onlyrises significantly far to the blue where the Cy3 absorbance is very lowand FITC absorbance is high. In fact, the apparent rise in R_(a) around460-470 nm may be an artifact of the low absolute precision of thespectra in that region. From the above discussion, it is clear that theexcitation contrast available for Cy3 vs. Cy3.5 is too low to be useful.

[0157] d. The emission contrast ratio for Cy3/Cy3.5 rises abruptly below570 nm. At the 567 nm peak of the Cy3 fluorescence, the absolute valueof S_(a) is approximately 6. This, combined with the factor 2 in theexcitation contrast parameter, should just about meet the goal of a10-fold discrimination between Cy3 and Cy3.5. The Ealing 35-3722narrowband interference filter is suitable, although it does overlap theHg 577/579 line significantly. Any stray light from this line gettingthrough the 546DF10 filter is, however, expected to be well blocked bythe chosen dichroic, Omega 560DRLP02.

[0158] e. The inability to differentially excite Cy3 vs. Cy3.5 meanswasteful bleaching of Cy3.5 during imaging of Cy3.

[0159] Imaging Cy3.5

[0160] The following points summarize high contrast imaging with thisdye:

[0161] a. The excitation contrast ratio between Cy3.5 and Cy3 rises tovery high values beyond approximately 565 nm. This region includes thepeak of the Cy3.5 excitation spectrum.

[0162] b. The mercury arc line at 577/579 nm is almost exactlycoincident with the peak of the Cy3.5 absorbance. At this wavelength theexcitation contrast ratio is approximately 25, i.e., very strongselective excitation of Cy3.5 relative to Cy3 is possible. An idealfilter for this purpose is the Ealing 35-3763. Note that the highcontrast is mainly a consequence of the Hg line position, not the filterbandwidth.

[0163] c. At the Hg 577/579 excitation wavelength, the excitationcontrast ratio for Cy3.5 relative to Cy5 is also quite large (absolutevalue approximately 8.0).

[0164] d. The emission contrast parameter for Cy3.5 vs. Cy3 is small atall wavelengths where the Cy3.5 emission is usefully strong, i.e.,isolation of Cy3.5 from Cy3 must rely mainly on excitation contrast.

[0165] e. The emission contrast for Cy3.5 vs. Cy5 is also large over aconsiderable spectral interval (and rises to very high values below 640nm). This permits a fairly broadband filter to be used to image Cy3.5; asuitable element is the Omega 615DF45. Almost no bleadthrough of eitherCy3 or Cy5 into the Cy3.5 channel is expected with the above combinationof excitation and emission conditions.

[0166] f. The Omega 590DRLP02 is a suitable dichroic for this channel.

[0167] Imaging Cy5

[0168] The following points are relevant to imaging with this dye:

[0169] a. Beyond 600 nm, the excitation contrast ratio for Cy5 vs. Cy3.5gets very large, i.e., very high excitation contrast relative to Cy3.5is possible.

[0170] b. The placement of the Cy5 excitation is determined by theextinction ratio relative to Cy5.5 rather than Cy3.5. The Cy5/Cy5.5excitation contrast parameter peaks at 650 nm with a numerical value of2.25. Thus, analogous with the Cy3/Cy3.5 pair, excitation contrast forCy5/Cy5.5 is poor.

[0171] c. There is no Hg line available for exciting Cy5. Thus, with anarc source, the continuum must be used, analogously with FITCexcitation. The “official” filter for exciting Cy5 in this way is theOmega 640DF20, which will give an excitation contrast with Cy5 of about1.8.

[0172] d. A much brighter source for exciting Cy5 is the He—Ne laser(632.8 nm). It does not, however, improve excitation contrast vs. Cy5.5.

[0173] e. The emission contrast for Cy5 vs. Cy3.5 peaks at 673 nm, justto the red of the fluorescence intensity peak. The closest availablefilter is the Omega 660DF32 where the emission contrast ratio isapproximately 3.1. This compounds the very high excitation contrast forCy5 vs. Cy3.

[0174] f. The emission contrast for Cy5 vs. Cy5.5 goes to very largevalues' at wavelengths shorter than 675 nm. The Omega 660DF32 filter isideally set up to take full advantage of this. Unfortunately, it isuncomfortably close to the 640DF20 exciter, so some flare fromreflected/scattered excitation light is to be expected. Use of the He—Nelaser would remove this problem.

[0175] g. The best available dichroic beamsplitter for Cy5 imaging isthe Omega 645DRLP02, particularly if a He—Ne is used as the excitationsource.

[0176] Imaging Cy5.5

[0177] CY5.5 is the penultimate dye of the set, and is very wellseparated from Cy7. Thus, only its contrast relative to Cy5 need beconsidered in detail:

[0178] a. The contrast parameter Rb for the Cy5.5/Cy5 pair rises tolarge values to the red of 670 nm. Thus, it is possible to achieve veryhigh excitation contrast for this pair of fluors (analogously toCy3.5/Cy3).

[0179] b. As with Cy5, the Hg arc is a poor source for exciting Cy5.5.The best available source is a 680 nm diode-pumped frequency doubled YAGmicrolaser (Amoco), which coincides with the peak of the Cy5.5absorbance. At 680 nm, the excitation contrast ratio for CY5.5/Cy5 is5.1. A suitable excitation filter is the Ealing 35-4068.

[0180] c. The emission contrast ratio between Cy5.5 and Cy5 peaks at 705nm, approximately 3 nm to the red of the Cy5.5 intensity curve. Thenumerical value for S_(b) at this point is 4. If the Omega 700EFLPlongpass emission filter is used, a contrast ratio of approximately 3(averaged out to 800 nm) is expected. This, combined with the highexcitation contrast, makes imaging Cy5.5 very clean.

[0181] d. At the expense of slightly lower emission contrast (this wouldnot be significant) and some loss of intensity, a bandpass filter suchas the Ealing 35-6345 could be substituted for the Omega 700EFLP. Thepotential advantage would be reduction of the infra-red background.i.e., overall improved image contrast.

[0182] Imaging Cy7

[0183] Cy7 is the reddest dye of the set. The excitation and emissionspectra are well separated from Cy5.5, and are well matched to the Omega740DF25/770DRLPO2/780EFLP triplet. The Oriel 58895 is an appropriate IRblocker for Cy7.

[0184] Filters selected for imaging the DAPI, FITC, Cy3, Cy3.5, Cy5,Cy5.5, Cy7 fluor set are summarized in the Table 2 below. None of thesefilter sets correspond to the filter sets supplied by manufacturers ofconventional fluorescence microscopes as narrow band excitation andfluorescence detection is mandatory to achieve sufficient contrast.TABLE 2 Epicube Filter Configuration (for 75 W Xe Arc Source ExcitationEmission Bandpass Dichroic Bandpass Flour Filter Beamsplitter Filter IRBlocking DAPI Zeiss Zeiss Zeiss None 365 nm 395 nm >397 nm FITC OmegaOmega Omega BG38 455DF70 505DRLP02 530DF30 Cy3 Omega Omega Ealing BG38546DF10 560DRLP02 35-3722 Cy3.5 Ealing Omega Zeiss BG38 35-3763590DRLP02 630/30 Cy5 Omega Omega Omega Oriel 58893 640DF20 645DRLP02670DF32 Cy5.5 Ealing Omega Omega Oriel 58893 35-4068 DRLP02 700EFLP Cy7Omega Omega Omega Oriel 58895 740DF25 770DRLP02 780EFLP

[0185] Characteristics of the microscope system are described in detailby Ballard S. G. et al. (J. Histochem. Cytochem. 41:1755-1759 (1993),herein incorporated by reference). A high pressure 75W DC xenon arc(XBO) was used as an excitation source because of its approximatelyconstant spectral power distribution. A Zeiss Axioskop microscopeworkstation equipped with a cooled CCD camera (Photometrics CH250) wasemployed. The objective lens was a 63×1.25 NA Plan Neofluar which shouldbe “plan” and apochromatic with a high numerical aperture. The filtersets selected were able to discriminate between the six fluors with amaximum contrast (Table 2). To minimize the crosstalk betweenfluorophores, the filter sets were selected on the basis of maximumspectral discrimination rather than maximum photon throughput. Imageexposure times were varied to adjust for photon flux differences andflux excitation cross-sections. Narrow band excitation and fluorescencedetection is mandatory to achieve sufficient contrast. Appropriateexcitation and emission filter sets were used to optically discriminatedthese fluors (Table 2).

[0186] The combinatorial labeling strategy relies on accuratemeasurements of intensity values for each fluorophore. Critical featuresare accurate alignment of the different images, correction of chromaticaberrations, and specific quantitation of each fluorophore. Becausesimple manual image manipulation could not realize these demands newsoftware was developed in our lab. This program comprises the followingsteps in sequential order: (1) correction of the geometric image shift;(2) calculation of a DAPI segmentation mask; (3) for each combinatorialfluor, calculation and subtraction of background intensity values,calculation of a threshold value and creation of a segmentation mask;(4) use of this segmentation mask of each fluor to establish a “Boolean”signature of each probe; (5) for each chromosome, display of thechromosome material next to the DAPI image; (6) create a composite grayvalue image, where each labeled object is encoded with a unique grayvalue; (7) final presentation of the results using a look-up-table (LUT)that assigns each gray value a particular color

[0187] The above-described program was developed on the basis of animage analysis package (BDS-Image) implemented on a Macintosh Quadra900. Image shifts caused by optical and mechanical imperfections werecorrected by the alignment of the gravity center (center of mass) of asingle chromosome in each image according to a procedure described byWaggoner, A. et al. (Methods Cell Biol 30:449-478 (1989)) and modified(du Manoir, S. et al., Cytometry 9:4-9 (1995); du Manoir, S. et al.,Cytometry 9:21-49 (1995), all herein incorporated by reference). TheDAPI image was used to define the morphological boundary of eachchromosome. Accurate chromosome segmentation was achieved bypre-filtering the images through a top-hat filter (Smith, T. G., et al.,J. Neurosci Methods 26:75-81 (1988); modified in du Manoir, S. et al.,Cytometry 9:4-9 (1995); du Manoir, S. et al., Cytometry 9:21-49 (1995)).The mode of the gray level histogram of the top-hat filtered DAPI imagewas used as the threshold. For each fluor background was eliminated bysubtracting the mean interchrombsomal fluorescence intensity from theimage. The mean of the chromosomal fluorescence intensities was used tocalculate the threshold for the individual segmentation mask of eachfluor. Individual DNA targets were assigned distinct gray valuesdepending on the “Boolean” signature of each probe, i.e. combination offluors used to label this DNA probe. In a final step a look-up table wasused to assign each DNA target a pseudocolor depending on this grayvalue, for display.

[0188] Switching the filter sets in excitation and emission path as wellas the dichroic mirror was done manually, but a computer-controlledelectro-mechanical solution will allow automation of the procedure.

[0189] 3. Optical Combs

[0190] In an alternative embodiment, selective excitation of multiplefluors and analysis of fluorescence spectral signatures can be carriedout using dispersion optics rather than wavelength-selectivetransmission filters. Such optics may be used to create filters of anypassband characteristic, including short-pass, long-pass, singlebandpass and multiple bandpass functions. In this method, a dispersionelement (prism or grating) is used in conjunction with awavelength-selective spatial filter to create the desired spectralresponse. The combination is referred to herein as a “comb filter.”Using a comb filter, the spectral distribution of the exciting light maybe tailored for optimum simultaneous excitation of multiple fluors. Theinverse comb filter may also be used to selectively block from the CCDcamera only the wavelengths used for excitation: the remainingwavelength intervals (corresponding to the gaps between teeth of thecomb) are available for spectral analysis of the fluorescence emitted bythe fluors. This analysis constitutes the spectral signature.

[0191] 4. Interferometers

[0192] In lieu of using the optical filters described above, aninterferometer may be used in conjunction with an epi-fluorescentmicroscope. A light source for excitation of fluorescence that is eithercoherent (e.g. an Argon laser) or incoherent (e.g. a Mercury arc lamp)may be used. A Mercury-Xenon mixed gas arc lamp is preferred due to itsintense Mercury lines and broad Xenon visible and near-infraredcontinuum.

[0193] Although any of a variety of interferometer designs (such asMichelson interferometer) may be employed, the use of a Sagnacinterferometer is preferred. The Sagnac interferometer has a largeracceptance angle, greater entendue, and is less sensitive to alignment,vibration, and temperature variations than a similar Michelsoninterferometer.

[0194] The Sagnac interferometer is a common path interferometer. Aninterferometer consists of two or more interfering beams of light. In acommon path interferometer there are two beams each traveling the samepath but in opposite directions. The optical paths are produced byreflecting light through beamsplitters, for example.

[0195] Multiple beam interferometers operate by dividing the opticalenergy from a light source into two substantially equal beams of light.The two beams of light are combined after one is permitted to passthrough a sample and the interference pattern (the changes in intensityof the combined light caused by the interference of two beams) isdetected.

[0196] In the Sagnac interferometer, the light source is also dividedinto two substantially equal parts. Changing the angle of incidence oflight on the beamsplitter, (by rotation of the interferometer, orrotation of an optic, such as a galvanometer driven mirror within theinterferometer) causes the optical path length to be changed along oneoptical axis of the interferometer. This produces a fringe pattern alongone axis of the detector, for example a CCD detector. The other axis ofthe detector can sample gray scale. As the optical path length isscanned, by rotating the interferometer or a mirror, the fringe patternproduces an interferogram at each pixel. The Fourier Transform of thisinterferogram yields the spectrum of light falling on that pixel of theCCD. Thus, an advantage of the Sagnac interferometer is that it producesan optical path difference across an entire field of view, rather thanat a single point.

[0197] Disturbances, such as a small shift of one of the opticalelements, effect both beams in the same way, and hence have no effect onthe measurement. This mechanical stability also makes the interferometerrelatively insensitive to temperature changes as well. Thus, anotheradvantage of the common path interferometer is its intrinsic stability.Sagnac interferometers and their use are well known (see, for example,U.S. Pat. Nos. 3,924,952; 4,410,275; 4,529,312; 4,637,722; 4,671,658;4,687,330; 4,836,676 and 5,108,183).

[0198] In one implementation of a Sagnac interferometer (J. Bruce Rafertet al., “Monolithic Fourier-Transform imaging spectrometer”, AppliedOptics, November 1995), the acceptance angle of the interferometer isdetermined to be:

q=2n tan⁻¹(w/8a)

[0199] where w/a=tan 30°, w is the aperture width of the interferometer,a is the length of each leg, and n is the index of refraction of theinterferometer glass. The input beam to the interferometer need not becollimated.

[0200] The interference pattern or interferogram is most preferablydetected with a CCD camera (such as a Princeton Instruments frametransfer CCD camera) capable of 512×512 pixels or larger. Since theinterferogram in a Sagnac interferometer has an angular dependence, eachpixel of the CCD detector measures a small interval of theinterferogram. The fringe spacing of the interferogram is set such thata pixel on the CCD detector can adequately sample the interferogram. TheOptical Path Difference (OPD) that a pixel can span, in order toproperly sample the interferogram is given by the relation:

OPD _(pixel)=λ_(min)/4

[0201] where λ_(min) is the shortest wavelength in the spectrum to bemeasured by the interferometer. This OPD_(pixel) determines thetheoretical limit of the resolving power of the interferometer.

[0202] As the OPD is being changed by the rotating mirror, theinterferogram is being moved across the CCD detector, such that themaximum optical path difference is then given by the relation,

OPD _(max) =N(OPD _(pixel))

[0203] where N is the linear dimension of the CCD detector in pixels.Each angular displacement of the light incident on the interferometerbeamsplitter may then correspond to one or several OPD_(pixel's). And,in the case of a CCD detector, one frame of CCD data is required tosample this angular displacement.

[0204] Finally then, each pixel comprises an interferogram whichcontains within it information about the spectrum of light falling onthe pixel, the intensity of light falling on that pixel, and the x and ycoordinates of the pixel. The spectrum of light may be recovered fromthe interferogram by the use of a computational Fourier Transformalgorithm.

[0205] In practice, because of the limited dynamic range of CCD's,typically about 10,000:1, the light used to excite the fluorescence mustbe blocked from entering the interferometer. This excitation light isoften 10⁸ to 10¹² more intense than the fluorescence that is emittedfrom the sample. Without blocking by using optical filtering, thisexcitation light would saturate the CCD. However, this filter need onlybe fabricated so as to block the excitation, all other wavelengths maybe allowed to pass.

[0206] In one embodiment, ultra violet (UV) light is used to excite thefluorescent probes. The UV light may be easily blocked with a long passinterference filter allowing the visible and near-infrared colors topass through to the interferometer. This embodiment has the advantagethat UV will excite many of the fluorescent dyes currently in use. Thisembodiment also has the advantage that it will allow better than 90%transmittance of the visible fluorescence to the interferometer. Thedisadvantage of UV is that it photobleaches the dyes faster than visiblelight.

[0207] Both the input and the output lens of the interferometer arepreferably very high efficiency camera lenses, and do not significantlyeffect the efficiency of imaging. The focus of the image within theinterferometer is most preferably adjusted so as to be constant for thevariable powers of the zoom eyepiece, and thus a microscope having thecharacteristic of infinite image distance (such as Olympus AX70microscope) are preferred.

[0208] The above-described interferometer possesses certain advantagesover optical filters. One key advantage is that all the light emitted byfluorescence is theoretically available for detection, whereas thetransmittance of an interference filter is limited. Another advantage isthat since the filters do not have to be changed, there is no imageshift due to the non-parallelism of filters.

[0209] E. Uses of the Present Invention

[0210] The capacity of the FISH (Fluorescent In situ Hybridization)methods and reagents of the present invention to detect and analyzechromosomal abnormalities, such as translocations, inversions,duplications, etc. can be used for a large number of applications.

[0211] 1. Applications Relating to the Cytogenetic Diagnosis of GeneticDisease

[0212] Among the primary applications of the present invention is thecytogenetic diagnosis of genetic disease, such as the pre- or post-nataldiagnosis of disease, complex tumor karyotyping, the analysis of cryptictranslocations (especailly through the use of telomere-specific probes).It provides a novel method for automated chromosome identification andanalysis. A large number of diseases (prenatal disease, cancers(especially BRCA1 or BRCA2 associated breast cancer), leukemias, Down'sSyndrome, etc.) are characterized by rearrangements and otherchromosomal abnormalities that can be discerned using the methods of theinvention.

[0213] Chromosome karyotyping by conventional cytogenetic bandingmethods is both time consuming, expensive and not easily automated. Thedetection of recurring genetic changes in solid tumor tissues bykaryotyping are particularly problematic because of the difficulty inroutinely preparing metaphase spreads of sufficient quality and quantityand the complex nature of many of the chromosomal changes, which makemarker chromosome identification based solely on banding patternsextremely difficult. Indeed, attempts to automate karyotype analysisover the past twenty years (e.g., pattern matching, eigen analysis) havefailed because robust computer algorithms could not be developed toreliably decipher complex banding patterns, particularly those ofextensively rearranged chromosomes.

[0214] It has been proposed that the next generation of cytogenetictechniques would be far superior by using bands that are definedmolecularly by hybridization of probes or probe sets each labeled with adifferent color (Nederlof, P. M. et al., Cytometry 11:126-131 (1990);Nederlof, P. M. et al., Cytometry 13:839-845 (1992); Lengauer, C. etal., Hum Mol Genet 2:505-512 (1993)). This would provide a highversatility and would constitute a quantum leap well comparable to theintroduction of chromosome banding and high resolution analysis ofchromosomes in prometaphase. Advantages of the FISH karyotype are theinstant identification of the chromosomal origin of marker chromosomes,double-minutes and homology staining regions (“HSRs”). Even “poorquality” chromosome spreads can be evaluated. If desired, one coulddesign a probe set for particular applications or for particularclinical applications, e.g. hematologic diseases, pre- or postnataldiagnosis. The development of specific probe sets that stain particularregions of chromosomes (e.g. telomeric regions) for the identificationof cryptic translocations would overcome limitations of the wholechromosome painting probes. Similarly, such probes could be used togenerate multicolor “barcodes” on individual chromosomes therebyfacilitating the automated analysis of karyotype. Probes can also bedesigned that would be specific for a particular arm of a chromosome,thereby permitting a molecular characterization of translocationbreakpoints, hot spots of recombination, etc. Other applications wouldinclude rapid evolutionary studies, provided that the protocols formulticolor FISH on human chromosomes can be adjusted, as expected, forapplications on other species.

[0215] 2. Applications Relating to the Detection of Infectious Agents

[0216] The methods of the invention may also be used to assess thepresence or absence of infectious agents (treponema pallidum,rickettsia, borrelia, hepatitis virus, HIV, influenza virus, herpes,Group B streptococcus, diarrhea-causing agents, pathogens causing acutemeningitis, etc.) in tissue, or in blood or blood products. This can beaccomplished by employing labeled probes specific for such agents.Moreover, by employing serotype-specific probes, the methods of thepresent invention permit the rapid serotyping of such agents, or thedetermination of whether any such agents carry drug resistancedeterminants. The methods of the present invention may be used to assesschromosomal abnormalities caused by exposure to radiation (such aspersonnel exposed to the radioactivity of nuclear power plants).

[0217] The methods of the present invention may be used to quantitatemicroorganisms that are difficult to propagate (such as anaerobicmicroorganism is involved in periodontal disease). The methods of thepresent invention provide a means for the rapid diagnosis of acutebacterial meningitis. Just as one can employ serotype-specific probes toperform serological analysis, one can employ probes that are specific toparticular drug resistance determinants, and thereby rapidly determinenot only the presence and identity of an infectious agent, but also itssusceptibility or resistance to particular antibiotics.

[0218] 3. Additional Applications

[0219] The methods of the present invention further permit simultaneousmapping of a large number of different DNA probes. With this techniquethe analysis of chromosomal number and architecture in individual intactcells becomes accessible. Interphase cytogenetics is already possiblewith small region specific probes, e.g. YAC-clones. The accuracy of suchanalysis could be increased by a three dimensional analysis using alaser scanning microscope, or more preferably, a CCD camera system witha Z-axis stepping motor coupled with 3-dimensional (3-D) imagedeconvolution software. Suitable 3-D image deconvolution software isobtainable from Imstar Corp. (Paris, France) or Scanalytics, Inc.(Boston). In addition, the use of such scanning microscope systems wouldultimately allow one to visualize all whole-chromosome painting probes,and telomere- or centromere-specific probes sets, in interphase nucleiand questions relating to intranuclear chromosomal organization as afunction of developmental status, cell cycle or disease state could beaddressed. Different models about the chromosomal organization ininterphase nuclei could finally be explored. Although conventional laserscanning microscopes currently do not allow the excitation of some ofthe fluorophores used, other, more appropriate fluors or devices may beemployed including 3 dimensional deconvoluting imaging systems..

[0220] Extended to non-mitotic cells, the methods of the presentinvention enable one to examine chromosome architecture or quantitatethe chromosome contents of nuclei in single hybridization experiments.Questions relating to intranuclear chromosomal organization as afunction of developmental status, cell cycle or disease state canaccordingly be addressed. In addition, the ability to quantitativelyassess the levels of multiple mRNAs or proteins in a single cell or todetermine if they exhibit different intracellular distributions couldprove extremely useful in addressing a myriad of interesting biologicalquestions. The multiparametric imaging of the present invention does notmerely increases the throughput of information, it also makes moreefficient use of the biological material. Thus, it can reveal spatialand temporal correlations as well as mosaicisms that might otherwise bedifficult to establish reliably. Since the intracellular distribution ofmRNAs and protein is not known a priori to be spatially distinct, as inthe case for the intra-nuclear chromosome domains, it will not bepossible to use the combinatorial labeling strategies in theseexperiments. However, many mRNA and protein antigens can be spectrallyresolved and detected using a multiplex format with n fluors. Thus, forthe first time the intracellular distribution of oncoproteins or tumorsuppressor proteins can be determined within the same cellsimultaneously.

[0221] F. Automated Karyotypic Analyses

[0222] One aspect of the present invention relates to automated,preferably computer-facilitated, karyotypic analyses. As describedabove, in one embodiment of the invention, the chromosomes of aparticular karyotype are pseudo-colored to thereby facilitate theassignment of the chromosomes, or the recognition of translocations,deletions, etc. In one sub-embodiment thereof, the digitized images ofthe chromosomes may be stored in a computer-readable storage device(such as a magnetic or optical disk) to facilitate their comparison withother chromosomal images or their transmission and study. In thisregard, probes may be employed that are translocation specific orspecific to sub-chromosomal elements or regions, such that thepseudocolorized chromosomes -or chromosomal elements are displayed tothe investigator as chromosomal images depicting a cytogenetic bandingpattern (for example, the cytogenetic banding pattern of the metaphasechromosomes of the patient). The position and sizes of individual bandsis preferably digitized and stored so that an image of the chromosomemay be stored on a computer. Similarly, the precise position of anytranslocation or other karyotypic abnormality can be discerned andstored.

[0223] The methods of the present invention thus permit karyotypicanalyses to be conducted more widely and more accurately than waspreviously feasible. The present invention may thus be used tosystematically correlate karyotypic abnormalities with disease orconditions. For example, karyotypes of asymptomatic individuals can beobtained and evaluated in light of any subsequent illness (e.g., cancer,Alzheimer's disease, etc.) or condition (e.g., hypertension,atherosclerosis, etc.) in order to permit a correlation to be madebetween a patient's karyotype and his or her predisposition to differentdiseases and conditions. Similarly, karyotypes of individuals havingdiagnosed diseases or conditions can be obtained and evaluated in lightof the extent of any subsequent progression or remission of the diseaseor condition so as to permit a correlation to be made between aparticular karyotype and the future course of a disease or condition.

[0224] In a further sub-embodiment, a computer or other digital signalanalyzer may be employed to orient and arrange the chromosomal images aswell as assigning and identifying the chromosomes of the karyotype.Thus, a computer or other data processor will, upon assigning aparticular chromosome to a particular designation (for example, uponassigning that a particular chromosomal image is the image of thechromosome 7 of the karyotype being evaluated), group the assignedchromosome with its homologue (e.g., the second chromosome 7 of thepatient's karyotype) and generate, via a printer, monitor, or otheroutput means, an ordered array of chromosomal images (such as one inwhich each autosomal chromosome is paired with its homologue, and inwhich the sex chromosomes X and Y are paired together).

[0225] In one sub-embodiment, the chromosomal images of such arrays willbe the pseudocolorized images discussed above. Alternatively, suchpsudocoloring may be internal to the process of assigning chromosomalidentity, and not displayed in the output of the computer or digitalsignal analyzer. Rather, in this sub-embodiment, the output generatedwill be the light-microscope visible cytogenetic banding pattern of themetaphase chromosomes of the patient whose karyotype is being evaluated.In a further sub-embodiment, a scale (in Morgans or other suitableunits) will be superimposed upon the chromosomal images.

[0226] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLE 1

[0227] Combinatorial Labeling of Chromosomes

[0228] In order to test the feasibility to produce 24 colors chromosomepainting probes representing the 22 autosomes and the two sexchromosomes were used. The DNA probes used were generated bymicrodissection.

[0229] Microdissected probes (National Center for Human Genome Research,Bethesda, Md.) give a very uniform labeling of the target region. Thedetailed protocols for microdissection and PCR amplification aredescribed by Telenius et al. (Telenius, H. et al., Genes, Chromosomes &Cancer 4:257-263 (1992); Telenius, H. et al., Genomics 13:718-725(1992); Meltzer, P. S. et al., Nature Genetics 1:24-28 (1992); Guan, X.Y. et al., Hum Mol Genet 2:1117-1121 (1993); Guan, X. Y. et al.,Genomics 22:101-107 (1994); Guan, X. Y. et al., Hum Genetics 95:637-640(1995), all herein incorporated by reference). For some chromosomesdifferent DNA-probes for the p- and the q-arms were available, namely 2,4, 5, 10, 11, 16, 18, and Y. For all other chromosomes microdissectedprobes painting the entire chromosome were used.

[0230] The first member of the set of preferred fluors, DAPI, was usedas a general DNA counterstain. The remaining fluors: fluorescein, Cy3,Cy3.5 (emission and excitation spectra are between Cy3 and Cy5), Cy5(Mujumdar, R. B. et al., Cytometry 10: 11-19 (1989), and Cy7 (emittingto the red of Cy5 (Ernst, L. A., et al., Cytometry 10:3-10 (1989)), wereused to combinatorially label different probes (Table 3). Distinctivefeatures of these dyes are high extinction coefficients, quantum yields,and photostabilities. Fluorescein is a xanthene dye with an extinctioncoefficient around 70,000 L/mol cm and quantum yields in optimal buffersaround 0.7. The respective values for cyanines are 1-200,000 and 0.3(Waggoner, A., Methods in Enzymology 246:362-373 (1995)).

[0231] After microdisection, the probes were subjected to a PCRamplification and labeled by nick translation. Fluorescein (Wiegant, J.et al., Nuc Acids Res 19:3237-3241 (1991)), Cy3, and Cy5 were directlylinked to DUTP for direct labeling. Cy3.5 and Cy7 were available asavidin or anti-digoxin conjugates for secondary detection ofbiotinylated or digoxinigated probes. They were synthesized usingconventional N-succinamide ester coupling chemistry. For each probe oneto three separate nick translation reactions were necessary, each with asingle labeled fluor-labeled triphosphate or biotin or digoxigenin(Table 3). TABLE 3 Fluor Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 FITC X XX X Cy3 X X X X X Cy3.5 X X X X X Cy5 X X X X Cy7 X X X X 13 14 15 16 1718 19 20 21 22 X Y FITC X X X X X X X Cy3 X X X X X Cy3.5 X X X X X XCy5 X X X X X X Cy7 X X X X X X

[0232] As expected probes labeled with equal amounts of different fluorsdid not give equivalent signal intensities for each fluor reflecting thefact that the filter sets were selected to maximize spectraldiscrimination rather than photon throughput. In order to diminishsignal intensity-differentials, probe concentrations for thehybridization mix had to be established carefully in a large number ofcontrol experiments. Hybridization conditions were optimized for thesemultiplex probes. Thus, probes were denatured and hybridized for two tothree nights at 37° C. to metaphase chromosome spreads in a conventional50% formamide hybridization cocktail. The slides were washed at 45° C.in 50% formamide/2×SSC three times followed by three washes at 60° C. in0.1×SSC to remove excess probe. After a blocking step in 4×SSC/3% bovineserum albumin for 30 min at 37° C. the biotinylated probes were detectedwith avidin Cy3.5 and the dig-labeled probes with anti-dig-Cy7.Fluorescein-dUTP, Cy3-dUT'P, and Cy5-dUTP did not require anyimmunological detection step. After final washes at 45° C. with4×SSC/0.1% Tween 20 three times, mounting medium and a coverslip wereapplied and the hybridization signals from each fluor imaged using thefilters sets listed in Table 3.

[0233]FIG. 1 provides a schematic illustration of the CCD camera andmicroscope employed in accordance with the present methods.

[0234]FIG. 2 shows the raw data from a karyotypic analysis ofchromosomes from a bone marrow patient (BM2486). Adjacent to each sourceimage is a chromosome “mask” generated by the software program. In FIG.2, panels A and B are the DAPI image and mask; panels C and D are FITCimage and mask; panels E and F are Cy3 image and mask; panels G and Hare Cy3.5 image and mask; panels I and J are Cy5 image and mask; andpanels K and L are Cy7 image and mask.

[0235]FIGS. 3A and 3B show the identification of individual chromosomesby spectral signature. FIG. 3A is the same photograph as FIG. 2, exceptthat it is gray scale pseudocolored. FIG. 3B displays the karyotypicarray of the chromosomes. The exceptional power of the methods of thepresent invention are illustrated by the ease with which thetranslocation of chromosomes 5 and 8 are identified in FIGS. 3A and 3B,relative to conventional non-chromosome specific karyotype analysis.

[0236] The above experiment demonstrates that five fluors can bespectrally discriminated to produce at least twenty four differentcolors. The combinatorial labeling schemes need not be as complex aspreviously thought, because using 5 fluors for probe labeling, only 9painting probes need to be labeled with as many as three fluors. A sixthfluor for probe labeling would allow up to 63 possible fluorcombinations. Such a high number of different targets will not berequired for most applications, but would allow the selection ofcombinations with the best spectral signature. These above-describedprotocols allow highly reliable and reproducible multicolor FISH.

EXAMPLE 2

[0237] Rapid Analysis of Multiple Oral Bacteria in Mixed CellPopulations Using Multiparametric Fluorescence In situ Hybridization(M-FISH)

[0238] In situ hybridization has been recognized as having potentialapplication as a means of analyzing individual bacterial cells in theabsence of culture. DeLong, E. F. et al. (Science 243:1360-1363 (1989)),for example, used oligonucleotide probes complementary to the 16sribosomal RNA (rRNA) sequences to differentiate an archaebacterium(Methanosarcina acetivorans) from the eubacteria Bacillus megaterium andProteus vulgaris. Van Den Berg, F. M. et al. (J. Clin. Pathol.42:995-1000 (1989)) used total genomic DNA to detect Campylobacterpylori in stomach tissue. Amann, R. et al. (Nature 351:161-164 (1991))detected Holospora obtusa in the macronucleus of the protozoanParamecium caudatum.

[0239] Prior to the present invention, however, the use of in situhybridization to differentiate microorganisms in clinical specimens withmixed populations, especially in a quantitative fashion, was difficultand time consuming. This is particularly true of bacteria withfastidious growth requirements, in which culture methods may result inthe selective enrichment of a subset of the organisms present in thespecimen.

[0240] A. Analysis of Non-Cross Hybridizing Microbial Species

[0241] To further illustrate the present invention, the M-FISH methodsof the present invention are used used to speciate bacteria involved inthe microbial etiology of periodontal disease.

[0242] Several methods have previously been used to establish themicrobial etiology of periodontal disease. Well over 200 bacterialspecies can be identified in one sublingual plaque sample usinglabor-intensive cultural methods (Dzink, J. L. et al., J. Clin.Periodont. 12:648-659 (1985); Moore, W. E. C.; J. Periodont. Res.22:335-341 (1987); Moore, W. E. C., Infect. Immunity 38:651-667 (1987)).This diversity has hampered the association of specific microbes in theetiology of the various forms and stages of inflammatory periodontaldiseases. Direct assessment, both qualitatively and quantitatively, ofplaque bacteria would obviate the need for culturing and its attendantdifficulties. DNA probes (French, C. K. et al., Oral Microbiol. Immunol.2:58-62 (1986)) have been widely used in a blot hybridization format asa direct method for bacterial assessment. However, this method does notallow for the direct visualization of the bacterial or provideinformation on the relative abundance of a particular bacterial within acomplex mixture.

[0243] In order to more simply illustrate the capacity of the inventionto speciate microbial strains, a test for in situ hybridizationspecificity is conducted using a mixture of bacteria that aremorphologically distinct and non-cross hybridizing. Using such amixture, one can immediately differentiate positive hybridization fromthat of non-specific binding by correlating the correct morphology withthe correct probe.

[0244] Thus, Fusobacterium nucleatum (25586), Porphyromonas gingivalis(33277), Eikenella corrodens (23834), Prevotella intermedia (25611), andActinobacillus actinomycetemcomitants (29522) were obtained from theAmerican Type Culture Collection, Bethesda, Md., and Capnocytophagaspecies, C. ochracea (C25) and C. gingivalis (DR2001) were obtained fromThe National Institute of Dental Research, Bethesda, Md.

[0245] Total genomic DNAs were isolated from F. nucleatum, P.gingivalis, E. corrodens, P. intermedia, A. actinomycetemcomitans, C.gingivalis, and C. ochracea. using the procedure of Chassy, B. M. etal., Appl. Environ Microbiol. 39:153-158 (1980)) as modified byDonkersloot, J. A. et al., J. Bacteriol. 162:1075-1078 (1985)). Each DNAwas labeled with ³²P and tested for crosshybridization with each of theother DNAs by dot blot hybridization. In all cases, the total genomicDNA probes show specificity for the bacteria from which it was derived;no hybridization with any of the other bacterial DNAs was detected.Since the genomic DNA probes were highly specific in the dot blotassays, they were used directly for the in situ identification of thesebacteria in reconstructed mixtures.

[0246] Total genomic bacterial DNAs are labeled by nick translationusing biotin-11-dUTP (BIO), digoxigenin-11-dUTP (DIG) ordinitrophenol-11-dUTP (DNP) as described by Lichter, P. et al., Hum.Genet. 80:224-234 (1988), herein incorporated by reference).Unincorporated nucleotides are removed using a Sephadex G-50 spin columnequilibrated with 10 mM Tris-HCI/1 mM EDTA/0.1% SDS, pH 8.0. LabeledDNAs (2-6 μg) are ethanol precipitated and redissolved in 100% deionizedformamide. F. nucleatum DNA is labeled with BIO-11-dUTP (BIO) anddetected with Cascade Blue avidin; A. actinomycetemcomitans DNA islabeled with DNP-11-UTP (DNP) and detected indirectly with an FITCconjugated antibody; and E. corrodens DNA is labeled with DIG-11-dUTP(DIG) and detected with a Rhodamine labeled anti-DIG antibody.

[0247] To prepare slides for in situ hybridization, small aliquots (1-2ml) of cultured bacteria are centrifuged at 1,200×g in an HBImicrocentrifuge. The cell pellet is suspended in phosphate bufferedsaline (PBS) and adjusted to approximately 10³ bacterial per μl.Aliquots of both pure cultures and synthetic mixtures are covalentlybound to activated glass slides prepared as described by Maples, J. A.(Amer. J. Clin. Pathol. 83:356-363 (1985), herein incorporated byreference). Alternatively, bacterial are spotted directly ontopositively (+) charged slides and air dried. Suitable slides arecommercially available from Fisher Scientific, Pittsburgh, Pa. (cat.#12-550-15), and retain all the bacterial strains listed abovethroughout the in situ hybridization procedure with high efficiency. Allslides are then fixed in Carnoy's B Fixative (Ethanol: Chloroform:Acetic Acid, 6:3:1) for 5 minutes.

[0248] The bacteria are hybridized simultaneously with the threedifferentially labeled genomic DNAs. Each sample is hybridized using 15μl of hybridization solution which contains 50 ng of labeled probe andDNAse-treated salmon sperm DNA (15 μg) in 50% (vol/vol) deionizedformamide/2×SSC (0.3 M sodium chloride/0.03 M sodium citrate, pH 7.0)/5%dextran sulfate. The solution is applied to the sample, covered with acoverslip and sealed with rubber cement. Both bacterial DNA and labeledDNA probe are denatured by heating at 80° C. for 5-8 minutes in a oven.These steps are sufficient to permeabilize the bacteria for probeassessability.

[0249] The DNAs were allowed to reassociate by incubating the slides at37° C. for 18 hrs in a moist chamber. Posthybridization washings,blocking and detection were as described by Lichter et al. (7). Briefly,the slides were washed 3× in 50% formamide/2×SSC at 42° C. for 5minutes, and then washed 3× in 0.1×SSC at 60° C. for 5 minutes. Theslides were then incubated in a solution of 3% bovine serumalbumin/4×SSC (blocking solution) for 30 minutes at 37° C.

[0250] Biotinylated probes were detected using fluoresceinisothiocyanate-(FITC)-avidin DCS (Vector Laboratories, Burlingame,Calif., 5 μg/ml) Cascade Blue-avidin (Molecular Probes Inc., Eugene,Oreg., 10 μg/ml)) or Rhodamine-avidin (Boehringer Mannheim,Indianapolis, Ind., 10 μg/ml).). Digoxigenin-labeled probes weredetected using FITC or Rhodamine conjugated sheep anti-digoxigenin Fabfragments (Boehringer Mannheim, 2 μg/ml). Dinitrophenol labeled probewas detected by incubating with rat anti-DNP antibodies (Novagen,Madison, Wis., 1:500 dilution)) and then with goat anti-ratFITC-conjugated antibodies (Sigma, St. Louis, Mo., 1 μg/ml). In someexperiments, bacterial DNA was counterstained with4,6-diamidino-2-phenylindole (DAPI) at a concentration of 200 ng/ml.

[0251] For detection, the fluorochrome-conjugated antibodies or avidinare diluted into a solution of 4×SSC/1% BSA and 0.1% tween-20 (200μl/slide) and incubated with the sample at 37° C. for 30 minutes in thedark. The slides are then washed 3 times in 4×SSC/0.1% tween-20 at 42°C. prior to viewing via epifluorescence microscopy.

[0252] Epifluorescence microscopy is conducted using a Zeiss Axioskop-20wide-field microscope with a 63×NA 1.25 Plan Neofluar oil immersionobjective and a 50W high pressure mercury arc lamp. Images are projectedwith a Zeiss SFL-10 photo-eyepiece onto a cooled charged-coupled device(CCD) camera (Photometrics CH220; 512×512 pixel array). Effectivemagnification is set by changing the microscope-camera distance, using abellows. The 8-bit greyscale images are recorded sequentially usingDAPI, FITC and Rhodamine filter sets (manufactured by C. Zeiss, Inc.,Germany) to minimize image offsets. Camera control, greyscale imageacquisition and image pre-processing are done using an Apple Macintosh11× computer preferably running custom software (Dr. Marshall Long; YaleUniversity). Images are pseudocolored, aligned and merged using aMacintoch 11 ci computer equipped with an accelerated 24-bit graphicssystem (SuperMac Spectrum 24 PDQ). The merging software, “Gene Join MaxPix” (Yale University) is preferably employed. This software assigns auser definable pseudocolor and greyscale value to each of the sourceimages, and then generates an output (“merged”) image based on the mostintense source image at each pixel location. Thus, objects with higherscaled brightness (e.g., probe signals) override the DAPI counterstainsignals in the corresponding image location. Objects displayed in mergedimage retain the color assigned to them and therefore appear distinctand easily identifiable. Merged images were photographed directly fromthe computer monitor.

[0253] A mixed sample of F. nucleatum, E. corrodens and A.actinomycetemcomitans, in a 1:1:1 ratio was used as a test sample. Ascan be seen in FIG. 4, the three bacteria are distinguishable on thebasis of morphology when stained with DAPI.

[0254]FIG. 4 shows the differentiation of bacteria by in situhybridization. Panels A-E represent in situ hybridization assay resultson a laboratory derived mixture of three bacteria (F. nucleatum, A.actinomycetemcomitans and E. corrodens) using a mixture of genomic DNAprobes for each organism. Panel A shows all the bacteria present in themicroscope field detected by (DAPI), a general DNA binding fluorophore.F. nucleatum is seen using Cascade Blue detection (panel B), A.actinomycetemcomitans with FITC detection (panel C) and E. corrodensusing Rhodamine detection (panel D). Panel E is a computer-mergedcomposite of panels B-D showing the differentiation of each bacteria inthe sample. Note that a single A. actinomyetemcomitans cell is detectedin a clump of E. corrodens (panel E) that could not be identified byDAPI staining (panel A). Panel F is a similar analysis of a mixture ofC. gingivalis (Rhodamine) and P. intermedia (FITC) when hybridized witha combination of genomic DNA probes for those organisms. Panel G showsan in situ hybridization assay for a combination of seven differentbacteria hybridized with a mixture of seven genomic DNA probes. F.nucleatum (1), E. corrodens (2) and A. actinomycetemcomitans (3) areshown in blue (Cascade Blue detection). C. gingivalis (4) and C.ochracea (5) are shown in red (Rhodamine detection), whereas P.intermedia (6) and C. gingivalis (7) are seen in yellow (FITCdetection). Phase contrast microscopy was used to assist in determiningmorphological differences between E. corrodens and A.actinomycetemcomitans which were not readily evident through thehybridization generated signals. Panel H represents an in situhybridization assay for the presence of A. actinomycetemcomitans (FITCdetection) in a plaque sample obtained from a patient with localizedjuvenile periodontist.

[0255] When assaying for Cascade Blue, only the F. nucleatum bacteria(FIG. 4, panel B) were seen, when assaying for FITC only A.actinomycetemcomitans bacteria (FIG. 4, panel C) were visible, and whenassaying for Rhodamine, only the E. corrodens bacteria (FIG. 4, panel D)were positive. The three separate fluorophore images were then merged togenerate the composite image that is shown in FIG. 4, panel E. Each ofthe morphologically distinct bacteria exhibit the correct fluorescencespecificity.

[0256] The four additional bacterial species are included in subsequentexperiments. These bacteria are combined pairwise such that each mixtureagain would differ morphologically. FIG. 4, panel F shows a combinedanalysis of P. intermedia (BIO label and FITC detection) and C.gingivalis (DIG label and Rhodamine detection). An analysis of C.ochracea and P. gingivalis, whose morphology is similar to the P.intermedia and C. ochracea and P. gingivalis, whose morphology issimilar to the P. intermedia and C. gingivalis pair, is also performed.In both cases, absolute specificity of the probe for its cognatebacteria is demonstrated.

[0257] Since the above experiments indicate that none of the bacteriacross-hybridized and that each species of bacteria, as grouped, could bedistinguished by morphology as well as by hybridization via the bindingof a specific fluorochrome, it was reasoned that all seven bacteriacould be detected in a single mixed sample. The experimental design wasto label the DNA from the bacteria within each of the threemorphologically distinct groups with the same “reporter” (BIO, DIG orDNP). The three groups would be initially differentiated byhybridization and the specificity of the fluorescence signal. Thebacteria within a group could then be differentiated on the basis ofmorphology.

[0258] To demonstrate this capacity of the present invention, P.intermedia and C. gingivalis DNAs were labeled with DIG, F. nucleatum,A. actinomycetemcomitans, and E. corrodens DNAs were labeled with BIOwhereas P. gingivalis and C. ochracea DNAs were labeled with DNP. Allseven probes were then combined and used for in situ hybridization onmixed bacterial samples containing all organisms (FIG. 4, panel G). InFIG. 4, panel G, the F. nucleatum, E. corodens, A. actinomycetemcomitansgroup is shown using Cascade Blue (BIO detection) and its members arenumbered 1, 2 and 3 respectively; the P. gingivalis and C. ochraceagroup is seen with Rhodamine (DIG detection) and these bacteria arenumbered 4 and 5 respectively; the C. gingivalis, P. intermedia group isidentified with FITC (DNP detection) and they are numbered 6 and 7respectively. All seven bacteria are clearly differentiated using thecombination of hybridization fluorescence and morphology. Whilemorphological identification could be made simply on the basis of size,shape and color of the hybridization signal, more definitivemorphological characterization is made by examination of the sampleusing phase-contrast optics.

[0259] To determine how few hybridization-positive bacteria could bedetected in a sample, 4.0 μl of E. corrodens containing 10⁵ bacteria/μlis mixed with 1.0 μl of serial dilutions of A. actinomycetemcomitans andthe 5 μl mixture applied to slides. The sample is then tested for thepresence of A. actinomycetemcomitans using a BIO-labeled probe for thatbacterium. Following the hybridization, the total bacteria on the slideare visualized using Propidium Iodide and the hybridization-positivebacteria are detected using Avidin-FITC. The results of this experimentshow that single A. actinomycetemcomitans cells could be detected in thepresence of an excess of at least 10⁴ E. corrodens cells. Thus, it isapparent that hybridization positive bacteria can be identified readilyeven when they constitute only a minor fraction of a mixed cellpopulation.

[0260] Clinical samples are not usually as free of contaminating debrisas laboratory derived mixtures of bacteria. To test the potentialutility of M-FISH for the analysis of clinical specimens, a dentalplaque sample from a patient with localized juvenile periodontitis, inwhich prior studies (Slots, J., Dent. Res. 84:1 (1976); Slots, J. etal., J. Clin Periodontal. 13:570-577 (1986); Zambon, J. et al., J.Periodontal. 54:707 (1983)) had suggested the involvement of A.actinomycetemcomitans as the microbial pathogen is employed. FIG. 4,panel H shows the results of an in situ hybridization experiment withthis patient's sample using BIO-labeled A. actinomycetemcomitans DNA anddetecting with FITC-conjugated avidin. As can be seen, a large number ofA. actinomycetemcomitans or other closely related organisms weredetected. Quantitative studies indicate that more than 50% of theindividually identifiable bacteria seen in the specimen hybridized withthe A. actinomycetemcomitans DNA probe. Cultural studies on this sampleshowed the presence of A. actinomycetemcomitans.

[0261] All of the above experiments employ a standard overnighthybridization time (˜18 hours) to differentiate the bacteria ofinterest. Even though such analyses can often be done faster than thetraditional methods of culturing and biochemical testing, a number ofexperimental parameters could be adjusted to give a more rapid assay. Itis well known that hybridization time is directly related to probeconcentration. Thus, by increasing probe concentration, hybridizationtime can be proportionately reduced. A mixture of F. nucleatum and A.actinomycetemcomitans is therefore analyzed using a 10-fold higherconcentration of probe than previously used (from 50 ng to 0.5 μg perslide). At the higher probe concentration no specific or non-specifichybridization is observed with undenatured probe and bacterial DNA.However, when the probe and bacterial DNA are denatured simultaneously,and the washing cycles begun immediately, some of the DNAs hybridizedduring the time necessary for subsequent sample processing.

[0262] In this experiment, in which 0.5 μg of A. actinomycetemcomitansprobe is introduced to a sample containing both A. actinomycetemcomitansand F. nucleatum bacteria using a 2.5 minute incubation at 37° C. priorto initiating the washing cycles, the hybridization signal is as strongand as specific as that observable in experiments utilizing 50 ng ofprobe and hybridizing for 18 hours. Furthermore, by reducing theblocking and antibody detection times to 20 minutes each, a total assaytime of 1.5 hours is achieved. Further reduction in assay times clearlycan be obtained using probe DNA directly labeled with the fluorophoredetector.

[0263] The above experiments provide a relatively rapid and simplemethod for identifying individual bacteria in mixed microbialpopulations using M-FISH. The method provides a quantitative assessmentof specific microorganisms without the necessity of culturing. Thetechnique can be applied to samples containing only a limited number oforganisms and a single hybridization-positive bacteria can be detectedquite easily in a 10⁴⁻ fold excess of hybridization-negative bacteria.In addition, as exemplified by the overlapping and “hidden” A.actinomycetemcomitans cell among the group of E. corrodens cells (FIG.4, panel E), bacterial specified that are not clearly identified by anon-specific analysis (i.e., DAPI staining of DNA, FIG. 4, panel A) canbe detected by the hybridization-specific assay. Since both thehybridization specificity and the morphological characteristics ofbacteria can be assessed simultaneously by this procedure, the number ofdifferent bacterial species can be increased still further by judiciousselection of probe labeling and fluorophore detector combinations.

[0264] However, morphological characteristics are not always a reliableindicator of bacterial identity since many different bacterial speciesappear morphologically similar and some are pleomorphic depending on theculturing methods. For example, A. actinomycetemcomitans are generallycoccobacillary in clinical specimens but can assume rod and evenfilamentous forms upon in vitro passage and in clinical specimens. Thus,additional fluorescence colors would preferably be employed to expandthe number of different bacteria that can be analyzed simultaneously andidentified unequivocally.

[0265] The intensity of the hybridization signal is extremely strongusing either whole genomic DNA or subtracted (suppressed) probes.Although data is collected by digital imaging, hybridization signals canbe detected readily by eye and recorded by conventional photographicmethods. However, the sensitivity and photon counting capabilities ofCCD-camera based imaging system offer considerable advantages forfurther refining microbial analysis by in situ hybridization. Detectionof single copy sequences for specific toxin or drug resistance genes inindividual bacteria should be feasible; individual human and murinegenes have been visualized, both in metaphase chromosomes and interphasenuclei by FISH (Lichter, P. et al., Hum. Genet. 80:224-234 (1988)). Thelevel of cross-homology between bacterial strains also can be rapidlyassessed by quantitating the photon output of the hybridization signalfrom individual bacteria in a specimen. Finally, and most importantly,digital imaging is essential to fully exploit the potential ofcombinatorial fluorescence.

[0266] As exemplified here, total genomic DNA can often provideadequately specific probes, thus eliminating the usual cloning andscreening efforts necessary for probe production. Although not requiredin these studies because of the absence of cross hybridization betweenthe bacterial genomes, suppression hybridization techniques (Lichter, P.et al., Hum. Genet. 80:224-234 (1988)) can be used to squelchcross-hybridization between organisms that share partial sequencehomology. For example, C. orchracea and C. sputigena exhibit 22-23%sequence homology (Zambon, J. et al., J. Periodontal. 54:707 (1983)) yetspecific hybridization to C. orchracea can be obtained with genomic DNAby preannealing the C. ochracea probe with an excess of C. sputigena DNAjust prior to the in situ hybridization reaction.

[0267] In sum, total genomic DNAs from seven anaerobic and facultativeoral bacteria recognized as pathogens in periodontal disease(Fusobacterium nucleatum, Porphyromonas (Bacteroides) gingivalis,Prevotella intermedia, Eikenella corrodens, Actinobacillusactinomycetemcomitans, Capnocytophaga gingivalis and Capnocytophagaochracea) are labeled non-isotopically and hybridized in situ toreconstructed bacterial mixtures. Each probe is hybridized uniquely tothe bacterium from which it was derived. Three bacterial strains aredifferentiated simultaneously by labeling DNA with different reportergroups and visualizing the hybridization signal with reporter-specificdetector proteins labeled with different fluorophores. By assessingbacterial morphology in conjunction with the hybridization signal eachbacteria in a mixture of all seven organisms can be identified. Thetotal assay time is as short as 1.5 hours. A dental plaque sample from apatient with localized juvenile periodontitis, an oral mixed microbialinfection, reveals numerous hybridization positive bacteria when probedwith DNA from A. actinomycetemcomitans, the putative microbial pathogenfor this disorder. This example demonstrates the usefulness of the insitu hybridization methods of the present invention for theidentification of individual non-cross hybridizing bacteria in mixedcell populations.

[0268] The seven bacterial species analyzed above are facultative orobligate anaerobes that are often found in association with inflammatoryperiodontal disease. The role of these microorganisms is uncertain dueto the lack of rapid and reliable methods to identify specifies. Directanalysis of oral microbial specimens circumvents biases imposed byculture methods and may allow a better understanding of the role ofthese bacteria in the pathogenisis of periodontal disease. Data on thedetection of A. actinomycetemcomitans in a specimen from a patient withlocalized juvenile periodontitis indicates that the above-described insitu hybridization procedure facilitates such studies. It alsocomplements traditional culture methods for the identification of abroad spectrum of microorganisms, especially notoriously slow growingbacterial such as anaerobes or mycobacterium. For example, swabs takenfrom early culture plates may be streaked on a slide and assayed by insitu hybridization using probes for suspected pathogens of interest.

[0269] The present invention permits one to increase further the numberof simultaneously detectable bacteria without increasing the number ofavailable fluorophores by using combinatorial fluorescence imaging(Nederlof, P. M. et al., Cytometry 11:126-131 (1990)). By labeling asingle probe with two (or more) different reporter molecules, theresultant hybridized probe will be detected by more than one of thefluorescent detectors and the signal will appear in more than one of theseparate flurorescence images. When these separate pseudocolored imagesare merged into a composite image (as in FIG. 4 panels E, F and G),fluorescence signals appearing at the same site on two images will be“blended” and generate a new pseudocolor that is distinguishable fromeither of the originals. In this fashion, three fluorophores have beenused combinationally to visualize seven probes simultaneously, eachappearing as a distinct color (Ried, T. et al., Proc Natl Acad Sci (USA)89:1388-1392 (1992)). By applying this combinatorial fluorescenceimaging strategy to bacterial identification, one can obtain a distinctpseudocolor for each of the seven bacterial species analyzed above.

[0270] The above example demonstrates the feasibility of differentiatingmultiple bacterial species (such as those found in the periodontalmicroflora) using multiparametric fluorescence in situ hybridization(M-FISH) and digital imaging microscopy. By monitoring bothhybridization signal specificity and bacterial morphology, sevendifferent bacteria are readily and simultaneously distinguished in amixed population using only three distinct fluorophores. Such methodsare readily applicable to the detection of bacteria (as well as viruses,and/or lower. eukaryotes) that may be present in other clinical samples(including those that contain complex mixtures of microflora (e.g.,stool, saliva, throat swabs, sputum, vaginal secretions, etc.) and thosethat are normally (e.g., central spinal fluid (meningitis), blood(sepsis), etc.) or non-clinical samples (food products, waterreservoirs, ecosytems, etc.). Furthermore, by expanding the number ofspectrally resolvable fluors used in combinatorial fashion, the numberof different types of bacteria, viruses and/or lower eukaryotes that canbe analyzed in a mixed population can be markedly increased.

[0271] B. Analysis of Cross Hybridizing Microbial Species

[0272] As indicated above, using differentially labeled total genomicDNA probes, the in situ hybridization and digital imaging fluorescencemicroscopy methods of the present invention can simultaneouslydifferentiate seven microorganisms in a laboratory derived mixed sample.This technique provides both single cell detection sensitivity and theability to correlate hybridization signal specificity with microbialmorphology. The relative abundance of a specific organism in a complexmixture or bacteria, can be readily assessed and a single hybridizationpositive bacteria can be detected in an excess of 10⁴ other bacteria.Moreover, the assays can be completed in less than 1.5 hours. However,none of the genomic DNAs from the seven bacteria used in the above studyexhibited cross-hybridization.

[0273] To illustrate the capacity of the present invention todifferentiate among related (i.e., cross-hybridizing) microflora, astudy is performed using Capnocytophaga DF 1 strains: C. gingivalis(DR2001), C. sputigena (D4), and C. ochracea (C25) Such strains exhibitcross-hybridization (Rubin, S. J., Eur. J. Clin. Microbiol. 3:253-257(1984)). The strains are obtained from the NIH Institute of DentalResearch, Bethesda, Md. Bacterial DNA is isolated as described above.Probes are made as follows: total genomic bacterial DNAs is labeled bynick translation using biotin-11-dUTP (BIO), digoxigenin-11-dUTP (DIG)or dinitrophenol-11-dUTP (DNP) as described by Lichter, P. et al. (Hum.Genet. 80:224-234 (1988)). Unincorporated nucleotides are removed usinga Sephadex G-50 spin column equilibrated with 10 mM Tris-HCl/1 mMEDTA/0.1% SDS, pH 8.0. Labeled DNAs (2-6 μg) are ethanol precipitatedand redesolved in 100% deionized formamide.

[0274] The genus Capnocytophaga encompasses a group of fusiform,gram-negative, capnophilic, fermatative, gliding bacteria (Ledbetter, E.R. et al., Arch. Microbiol. 122:17-27 (1979)) that are commoninhabitants of the oropharyngeal flora (Holdeman, L. V. et al., J.Periodon. Res. 20:475-483 (1985)). C. gingivalis, C ochracea and C.sputigena have been implicated in gingivitis in children (Moore, W. E.C. et al., Infect. Immun. 46:1-6 (1984)) and periodontitis in juvenilediabetics and adults (Dzink, J. L et al., J. Clin. Periodont. 12:648-659(1985); Mashimo, P. A. et al., J. Periodont. 54:420-430 (1983); Slots,J. et al., J. Dent. Res. 63:414-421 (1984)). These three bacteria aremembers of the first of three groups of “Dysgonic Fermenters” (DF-1)defined by the Center for Disease Control (Speck, H. et al., Zbl. Bakt.Hyg. A. 266:390-402 (1987)). The DF-2 group contain species designatedas C. canimorsus and C. cynodeami, although these bacteria appear bothphenotypically and genetically distinct from the Capnocytophaga speciesof the DF-1 group (Brenner, D. J. et al., J. Clin. Microbiol. 27:231-235(1989)). Few of the microbes comprising the DF-3 group have beencharacterized, however it is thought that they are closely related tothe Capnocytophaga DF-1 species (Gill, V. J. et al., J. Clin. Microbiol.29:1589-1592 (1991)). Members from all three DF groups have beenidentified as the etiologic agents found in some cases of septicemia,joint infections and endocarditis (Arlet, G. et al., Ann. Biol. Clin.44: 373-379. (1986); Juhl, G. et al., J. Infect. Dis. 149:654 (1984);Matlow, A. et al., J. Infect. Dis. 152:223-234 (1985); Mosher, C. B. etal., J. Clin. Microb. 24:161-162 (1986); Parenti, D. M. et al., J.Infect. Dis. 151:140-147 (1985); Ratner, H., Clin Microbiol 6:10 (1984);Rummens, J. L. et al., J. Clin. Microbiol. 75:376 (1985); vonGraevenitz, A., Eur. J. Clin. Microbiol. 3:223-224 (1984)). Theincreased spectrum of oral and intra-oral infections caused byCapnocytophaga DF-1 species shows the pathogenic potential of this genusand may reflect differences in clinical significance of the respectivespecies in medically important infections and periodontal disease (vonGraevenitz, A., Eur. J. Clin. Microbiol. 3:223-224 (1984)). Conventionalmethods of identifying bacteria from oral sources requires first,isolation of single bacterial colonies from the oral microbial milieu,and second, the assignment of isolates to genus and species based uponcellular. morphology, gram stain, fermentation of carbohydrates andhydrolysis of various substrates. These procedures require considerabletime and expertise, taking upwards of 2 weeks to identify single speciesfrom clinical samples. Such conventional methods often fail to isolateCapnocytophaga from oral samples because these bacteria are slowgrowing, fastidious as to growth conditions and are therefore overgrownby other oral flora (Mashimo, P. A. et al., J. Periodont. 54:420-430(1983); Rummens, J. L. et al., J. Clin. Microbiol. 75:376 (1985)).Moreover, such methods may fail to discriminate C ochracea and C.sputigena unambiguously due to phenotypic similarities (Gill, V. J. etal., J. Clin. Microbiol. 29:1589-1592 (1991)).

[0275] Recent methods of microbial identification using enzymatic tests(Heltsberg, O. et al., Eur. J. Clin. Microbiol. 3:236-240 (1984);Kristiansen, J. E. et al., Eur. J. Clin. Microbiol.3:236-240 (1984)),specific immunologic or molecular probes (Murray, P. A. et al., OralMicrobiol. Immunol. 6:34-40 (1991); Smith, G. L. F. et al., OralMicrobiol. Immunol. 4:41-46 (1989)) have been used with some success foridentifying bacteria in clinical oral samples. With the exception ofenzymatic techniques, these methods have not been applied to theidentification of Capnocytophaga spp. Enzymatic tests still require theisolation of pure colonies of bacteria, whereas immunologic andmolecular methods can be performed directly on an oral sample, therebyovercoming the difficulties of isolating pure colonies of fastidiousmicroorganisms. Generally, molecular methods rely on the use of DNAprobes, formatted as a dot-blot hybridization assay, for theidentification of a particular microorganism in a clinical sample(Parenti, D. M. et al., J. Infect. Dis. 151:140-147 (1985)). A majordrawback of this method is that DNA from 10³ to 10⁶ microorganisms ofinterest are required for such assays, therefore bacteria in lowabundance in a mixed population are likely not to be detected. Inaddition, cross-hybridization, which is common in closely relatedspecies, can impede identification to the species level.

[0276] It had been previously reported (Williams, B. L. et al., Arch.Microbiol. 122:35-39 (1979)) that C. gingivalis, C. ochracea, and C.,sputigena DNAs exhibit up to 22% sequence homology, based onreassociation kinetics. Using conventional in situ hybridizationmethods, such extensive homology would appear to preclude the use oftotal genome DNA probes for the speciation of Capnocytophaga strains andnecessitate the production and characterization of species-specificsubgenomic clones. However, one of the attributes of the computerizeddigital imaging camera system of the present invention is the ability tocollect hybridization data in the form of quantitative fluorescenceimages and to process such image data with appropriate computersoftware.

[0277] The intensity of the hybridization signal (i.e., fluorescencephoton output) from a bacteria (“bacteria A”) A will be greatest with agenomic DNA derived from that bacteria. Related strains which share someDNA sequences in common with a DNA probe of genomic DNA for bacteria A(genoric DNA probe A) will give less intense hybridization signals,which directly reflect the extent of sequence homology. Unrelatedbacteria that share little or no sequence homology to bacteria A willyield virtually no fluorescence signal at all. Once the relative extentof DNA sequence homology between stains has been established,cross-hybridization noise can be electronically suppressed (preferablyvia appropriate computer software and image thresholding techniques)thus directly leading to bacterial identification.

[0278] Computer thresholding of fluorescence images is used to establishthe extent of cross-hybridization (i.e., sequence homology) between thethree Cagnocytophaga species. In this experiment, pure samples of eachbacteria are separately hybridized with biotinylated DNA probes from allthree species. For example, three slides are prepared that contain onlyC. ochracea cells. The first slide is hybridized in situ usingBIO-labeled C. ochracea genomic DNA, the second slide is hybridized insitu using BIO-labeled C. gingivalis genomic DNA and the third slide ishybridized in situ using BIO-labeled C. sputigena genomic DNA. Theslides are then washed and hybridization positive bacteria areidentified using FITC-avidin.

[0279]C. ochracea and C. gingivalis did not crosshybridize with oneanother, however C. sputigena cross-hybridized with both C. gingivalisand C. ochracea. A computer analysis of the intensity of the fluorescenthybridization signals indicates that C. sputigena had significantsequence homology with C. ochracea (˜22%) but much less (˜5%) with C.gingivalis.

[0280] To demonstrate how image thresholding can be used for bacterialspeciation, a slide with a mixture of C. sputigena and C. orchracea ishybridized with Bio-labeled genomic DNA from C. sputigena. To preparesuch slides for in situ hybridization, small aliquots (1-2 ml) ofcultured bacteria are centrifuged at 1,200×g in a HBI microcentrifuge.The cell pellet is suspended in phosphate buffered saline (PBS) andadjusted to approximately 10³ bacteria per μl. Aliquots of both purecultures and synthetic mixtures are spotted directly onto commerciallyavailable (Fisher Scientific, cat #12-550-15) positively (+) chargedslides and air dried. All slides are then fixed in Camoy's B Fixative(Ethanol: Chloroform: Acetic Acid, 6:3:1) for 5 minutes.

[0281] Hybridization is accomplished using 15 μl of hybridizationsolution which contained 50 ng of labeled probe and DNAse treated salmonsperm DNA (15 μg) in 50% (vol/vol) deionized formamide/2×SSC (0.3 Msodium chloride/0.03 M sodium citrate, pH 7.0)/5% dextran sulfate. Thesolution is applied to the sample, covered with a coverslip and sealedwith rubber cement. Both bacterial DNA and labeled DNA probe aredenatured by heating at 80° C. for 5-8 minutes in an oven. These stepsare sufficient to permeabilize the bacteria for probe assessability.

[0282] The DNAs are allowed to reassociate by incubating the slides at37° C. for 2.5 min. in a moist chamber. Posthybridization washings,blocking and detection are conducted as described by Lichter, P. et al.(Hum. Genet. 80:224-234 (1988)). Briefly, the slides are washed 3 timesin 50% formamide/2×SSC at 42° C. for 5 minutes and then washed 3 timesin 0.1×SSC at 60° C. for 5 minutes. The slides are then incubated in asolution of 3% bovine serum albumin/4×SSC (blocking solution) for 30minutes at 37° C.

[0283] Biotinylated probes are detected using fluoresceinisothiocyanate-(FITC)-avidin DCS (Vector Laboratories, 5 μg/ml), CascadeBlue-avidin (Molecular Probes Inc., 10 μg/ml) or Rhodamine-avidin(Boehringer Mannheim, 10 μg/ml). Digoxigenin-labeled probes are detectedusing FITC or Rhodamine conjugated sheep anti-digoxigenin Fab fragments(Boehringer Mannheim, 2 μg/ml). Dinitrophenol labeled probe is detectedby incubating with rat anti-DNP antibodies (Novagen, 1:500 dilution))and then with goat anti-rat FITC-conjugated antibody (Sigma, 1 μg/ml).In some experiments, bacterial DNA was counterstained with4,6-diamidino-2-phenylindole (DAPI) at a concentration of 200 ng/ml. Fordetection, fluorochrome-conjugated antibodies or avidin are diluted intoa solution of 4×SSC/1% BSA and 0.1% tween-20 (200 μl/slide) andincubated with the sample at 37° C. for 30 minutes in the dark. Theslides are then washed 3 times in 4×SSC/0.1% tween-20 at 42° C. prior toviewing.

[0284] Hybridization is visualized via epifluorescence microscopy usinga Zeiss Axioskop-20 wide-field microscope with a 63×NA 1.25 PlanNeofluar oil immersion objective and a 50W high pressure mercury arclamp. Images are projected with a Zeiss SFL-10 photo-eyepiece onto acooled charged-coupled device (CCD) camera (Photometries CH220; 512×512pixel array). Effective magnification is set by changing themicroscope-camera distance, using a bellows. The 8-bit greyscale imagesare recorded sequentially using DAPI, FITC and Rhodamine filter sets,manufactured by C. Zeiss, Inc., Germany to minimize image offsets.Camera control, greyscale image acquisition and image pre-processing aredone as described above.

[0285] All bacteria are found to be hybridization positive in thisexperiment, although some appear to fluorescence more intensely thanothers. After the fluorescence image had been thresholded (using theGene Join Max Pix software; Yale University) to remove components of theimage that exhibited less than 25% of the maximum fluorescenceintensity, only a subset of the original bacteria appear hybridizationpositive. These bacteria are all C. sputigena because only they exhibit100% homology to the probe. This general strategy can be used tospeciate multiple related microorganisms, provided the level of theirsequence relatedness is known so that appropriate thresholdingparameters can be applied. Bacteria exhibiting up to 40% sequencehomology can be discriminated with genomic DNA probes using thistechnique.

[0286] The Gene Join Max Pix software can be used in a second way toachieve bacterial speciation with genornic DNA probes. This embodimentexploits the fact that when separate fluorescence images (e.g.,fluorescein and rhodamine) are merged to form a composite image; thesource image with the greatest fluorescent intensity (i.e., photonoutput) at each pixel location will be color-dominant in the mergedimage. This attribute permits the use of multiple differentially labeledgenornic DNA probes simultaneously to probe a single or mixed bacterialpopulation. Since each bacteria in a mixture will exhibit the greatestfluorescence signal with the genomic probe that is 100% homologous, theimage merging process results in effective speciation.

[0287] This is demonstrated by the following experiment. A mixture ofthe three Capnocytophaga strains is hybridized using a 1:1:1 of allthree genomic DNA probes, each labeled with a different reporter. C.ochracea DNA was labeled with DNP-11-dUTP for detection with FITC(yellow green), C. gingivalis DNA was labeled with biotin-11-dUTP fordetection with Cascade Blue (blue) and C. sputigena DNA was labeled withDIG-11-dUTP for detection with Rhodamine (red). The image generated byCascade Blue shows intense signals for some bacteria present in thesample, presumably C. gingivalis cells and weak signals for otherbacteria. The FITC fluorescence image produced by the C. ochracea probeand the Rhodamine fluorescence imge produced by the C. sputigena probealso show both intense signals for some bacteria and weak signals forothers, reflecting interspecies hybridization. However, each bacteriumshows an intense signal in one of the three images and a weaker (orabsent) signal in the other images (reflecting interspecieshybridization). Accordingly, through the use of thresholding (i.e.,using the signal generated to determine which probe gave the moredominant signal with which bacteria), the method enables one todetermine the species of each bacterium.

[0288] Thus, in contrast to traditional methods of culturing and testingbiochemical products for the differentiation and/or speciation of abacteria, that are often difficult and time consuming, the in situhybridization methods of the present invention provide a rapid andsimple method for the speciation of cross-hybridizing rnicroorganisms,such as the oral Capnocytophaga. The basis of this method relies on thefinding that all microorganisms within different genera and species haveunique DNA sequences within their genome. Closely related organisms willhave some DNA sequences in common, and the amount of suchcross-hybridizing sequences will directly affect their ability to bedifferentiated by in situ hybridization.

[0289] In this analysis, the fluorescence images obtained from in situhybridization with each detector (e.g., Rhodamine, FITC or CascadeBlue), which reflect the amount of hybridization of a bacterium to eachof the genomic DNA probes, are digitalized via computer. If a bacteriahas no cross-hybridizing sequence with any of the other bacteria it willhybridize only to its homologous DNA and thereby be detected by only asingle detector and its presence will be found in only the imagecorresponding to that detector. However, if a bacteria does havecross-hybridizing sequences with one of the other bacteria, then it willbe detected by the DNA probes from each bacteria and its presence willbe seen in the corresponding images for each probe (detectors). However,the intensity of the signal generated in each image will be directlyproportional to the amount of hybridization of a bacterium with a DNAprobe. The corresponding images from each detector are pseudocolored andoverlaid (merged) to form a composite image. Since any given pixel onthe monitor can only be a single color, only the most intense signal ateach pixel is shown, and displayed as the corresponding pseudocolor.

[0290] Whereas two of the Capnocytophaga species (C gingivalis and Cochracea) of the above experiment show no cross-hybridization, the thirdspecies (C sputigena) cross-hybridized with both of the precedingorganisms. The amount of cross-hybridization as estimated by a computeranalysis of the intensity of the hybridization signal following in situhybridization is in good agreement with that found in the literature(22-23% for C. sputigena and C. ochracea and 9-11% for C. sputigena andC. gingivalis (Heltberg, O. et al., Eur. J. Clin. Microbiol. 3:236-240(1984)). The 22-23% crosshybridization of C. sputigena and C. ochraceais evident in the composite image within some of the C. sputigena cells,but not evident in the C. ochracea cells. This may reflect differencesin how the cross-hybridizing sequences are arranged within thosegenomes.

[0291] This example further demonstrates that for an in situhybridization analysis, the DNA probe need not be totally specific.Cross-hybridizing sequences must only be reduced below the levelnecessary for showing specificity. The level of unique sequencesnecessary for specificity would desirably be ascertained for eachbacteria of interest, since it would probably be affected by not onlythe total number of cross-hybridizing sequences, but also how thosesequences are arranged within a genome. However, some microorganisms,such as Neisseria meningitis and N. gonorrhoea show greater than 80%sequence homology (Kingsbury, D. T., J. Bacteriol. 94:870-874 (1967)).Such large amounts of cross-hybridizing sequences may result in anambiguous analysis using in situ hybridization with total genomic DNAprobes that can be addressed by the methods of the present invention.

[0292] Thus, in sum, multiparametric fluorescence in situ hybridization(M-FISH) and computer assisted digital imaging microscopy have been usedto develop a rapid method for differentiating C. gingivalis, C ochraceaand C. Sputigena with total genomic DNA probes. Although these bacteriaexhibit up to 22% sequence homology, cross-hybridization signals can becompletely suppressed. Speciation of these three Capnocytophaga speciescan be achieved in as little as 30-90 minutes, markedly faster than the2-3 weeks required using conventional microbiological methods. Thegeneral discrimination strategy reported here is applicable to the broadspectrum of microbial pathogens for which an extent of sequence homologyis known.

EXAMPLE 3

[0293] Synergistic Use of Nucleic Acid Amplification Technology inConcert with M-FISH Analysis

[0294] Nucleic acid amplification technology has greatly increased theability to investigate detailed questions about the genotype or thetranscriptional phenotype in small biological samples, and has providedthe impetus for many significant advances in biology, especially in thefield of genetics.

[0295] One aspect of the present invention concerns the use of theabove-described multiparametric fluorescence in situ hybridization(M-FISH) methods in concert with one or more methods of nucleic acidamplification to facilitate the detection and characterization ofmutations, chromosomal elements, chromosomal rearrangements, andinfectious agents (e.g., viruses, proviruses, etc.) that may be presentin small amount or proportion in a sample undergoing analysis. Forexample, through the use of fluorophore combinations designed formultiparametric color coding, a 6-fluor tagging approach permits thesimultaneous identification of 63 different types of signals by virtueof their unique spectral signatures.

[0296] As described above, the M-FISH method of the present inventioncan distinguish and identify all of the chromosomes of a humankaryotype. However, by amplifying a specific gene, allele or chromosomalelement, and employing one or more labeled probes that are capable ofspecifically hybridizing to the amplified sequences, the inventionpermits the determination of whether a particular karyotype contains thegene, allele or chromosomal element in question. By way of illustration,the use of a probe specific for a deletion, rearrangement, insertion orother mutation characteristic of a genetic disease (e.g., hemophilia,cystic fibrosis, breast or other cancer, etc.) it is possible todiagnose whether a patient exhibits the genetic disease (e.g., todiagnose cystic fibrosis, etc.). Similarly, the invention permits one todetermine whether a patient's chromosomes carry a recessive alleleassociated with genetic disease. Likewise, the invention permits one todetermine whether a patient is predisposed to a disease by virtue of thepresence of a genetic lesion (e.g., a mutation in the apoE, p53, rb, orbrcal1/2 genes) associated with a future disease state (such asAlzheimer's Disease, heart disease, cancer, etc.).

[0297] Similarly, as applied to the investigation of microbes (e.g.,bacteria, viruses, and lower eukaryotes), the M-FISH method of thepresent invention can distinguish and identify the species ofmicroorganisms present in a clinical or non-clinical sample. However, byamplifying a specific gene, allele or chromosomal element, and employingone or more labeled probes that are capable of specifically hybridizingto the amplified sequences, the invention permits the determination ofwhether a particular microorganism contains the specific gene, allele orchromosomal element. Thus, for example, by using a probe specific for anantibiotic resistance determinant, a cellular antigen, a toxin, etc.,the combined use of M-FISH and nucleic acid amplification permits adetermination of whether a particular microbial or viral strain isresistant to an antibiotic, or is pathogenic, or expresses a toxin. Suchcombination of methodologies thus permits the serotyping andsubspeciation of pathogens without any requirement of culturing and/orpurification.

[0298] The M-FISH methods of the present invention particularly concernprobe sets and methods that are sufficient to characterize both thegenotypic and phenotypic charateristics of micobes present in apreparation. A genotypic probe is one capable of specificallyhybridizing to phylogenetically related species of microbes;hybridization of such a probe to nucleic acid of a preparation thusreveals whether a preselected microbe, or its cross-hybridizing (i.e.,phylogenetically) related microbes are present in such a preparation. Aphenotypic probe is one capable of specifically hybridizing to genes, orgenetic elements associated with a particular phenotype (e.g.,antibiotic resistance, toxin production, antigen presentation, etc.).

[0299] The invention particularly contemplates the multiparametric useof multiple fluors, and particularly concerns the embodiments in which2, 3, 4, 5, 6 or more fluorophores are employed so as to permit thedetection and/or characterization of 3, 7, 15, 31, 63 or morecombinations of genotypic or phenotypic elements. Thus, for example, iffive fluors are employed, 31 genotypic/phenotypic elements can beprobed. Any combination of such elements can be employed. For example,25 of such elements can be genotypic elements (thus permitting theidentification of 25 different species of microbes), whereas 6 of suchelements can be genotypic (for example permitting the determination ofwhether any of the 25 identified species are resistant to any of 4antibiotics or present any of 2 toxins). Similarly, 10 of such elementscan be genotypic elements (thus permitting the identification of 10different species of microbes), whereas 21 of such elements can begenotypic (for example permitting the determination of whether any ofthe 10 identified species. are resistant to any of 9 antibiotics,present any of 3 toxins, present any of 4 surface antigens, and expressany of 5 genes).

[0300] Any of a wide group of bacteria (e.g., E. coli strains andstrains of other enterics (e.g., Salmonella), Clostridria, Vibrio,Corynebacteria, Listeria, Bacilli (especially B. anthracis),Staphylococcus Streptococci (especially beta-hemolytic Streptococci andS. pneumoniae), Borrelia, Mycobacterium (especially M. tuberculosi);Neisseria (especially N. gonorrhoeae), Trepanoma, bacteria implicated inperiodontal disease (e.g., Fusobacteria, Porphyromonas, Eikenella,Prevotella, Actinobacillus, and Capnocytophaga species), etc.), viruses(e.g., parvoviruses, papoviruses, herpesviruses, togaviruses,retroviruses (especially HIV), rhabdoviruses, influenza viruses, etc.),and lower eukaryotes (fungi (e.g., Dermatophytes; Pneumocystis,Trypanosoma; etc.), yeast, helminths, nematodes, etc.) can be detectedand characterized using such a method.

[0301] Significantly, the combined use of a nucleic acid amplificationmethod and the multiparametric fluorescence in situ hybridizationmethods of the present invention can be used to explore the quiescenceor expression state of cells. Thus, by employing probes specific to RNAproduced by, for example, growing cells, cells expressing tumor antigensor hormones, etc., the methods of the present invention can determinenot merely the presence of tumor cells, but the extent of theirmalignancy.. Such mRNA profiling may be conducted even in circumstancesin which standard cDNA hybridization approaches may not be sensitiveenough to detect changes in the concentration of low abundancy geneproducts.

[0302] While the combined use of a nucleic acid amplification method andthe multiparametric fluorescence in situ hybridization methods of thepresent invention can employ any suitable amplification method ormethods (e.g., Polymerase Chain Reaction (Mullis, K. et al., Cold SpringHarbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184;Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No.4,582,788; Saiki, R. et al., U.S. Pat. No. 4,683,194 and Higuchi, R.“PCR Technology,” Ehrlich, H. (ed.), Stockton Press, NY, 1989, pp61-68), Strand Displacement Amplification (Walker, G. T. et al., Proc.Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992); Walker et al., U.S. Pat.No. 5,270,184; Walker, U.S. Pat. No. 5,455,166); Ligase Chain Reaction(Segev, WO90/01069; Birkenmeyer, WO93/00447), etc.), the preferredmethod of amplification is the Rolling Circle Amplification (RCA) method(Caplan, M. et al., PCT Patent Application Publication No. WO 97/19193;herein incorporated by reference in its entirety).

[0303] Rolling Circle Amplification (RCA) is an amplification strategyin which nucleic acid amplification is driven by a DNA polymerase thatcan replicate circularized oligonucleotide probes with either linear orgeometric kinetics, under isothermal conditions. In detail, RCA involvesincubating at least one one rolling circle replication primer (RCRP)with at least one amplification target circle (ATC). The ATC comprises asingle stranded circular DNA molecule that contains a regioncomplementary to the RCRP, such that the RCRP can hybridize to the ATCand mediate amplification in the presence of a DNA polymerase.

[0304] In the presence of two primers, one hybridizing to the “+”strand, the other to the “−” strand of DNA, a complex pattern of DNAstrand displacement ensues that generates 10⁹ or more copies of eachcircle in 90 minutes, enabling detection of point mutations in humangenomic DNA. This expanding cascade of strand displacement andfragrnent-generation events is termed “DNA Hyperbranching,” and thespecial rolling circle amplification driven by two primers is termed“Hyperbranched-RCA” or “HRCA.” Using a single primer, RCA generateshundreds of tandemly linked copies of a covalently closed circle in afew minutes.

[0305] It is preferred to conduct HRCA using exo(−) Vent DNApolyrnerase, or the large fragment of Bst DNA polymerase (Aliotta, J. M.et al., Genet. Anal. 12:185-195 (1996); Thomas, D. C. et al., Clin.Chem. 43:2219 Abs 38 (1997); both herein incorporated by reference), andby the Sequenase 2.0 variant of T7 DNA polymerase. It had been shownthat Sequenase supports rolling circle amplification of circularoligonucleotides, albeit relatively slowly (Fire, A. et al., Proc. NatLAcad. Sci. (USA) 92:4641-4645 (1995); Liu, D. et al., J. Amer. Chem.Soc. 118:1587-1594 (1996)). The efficiency of RCA and HRCA reactions isincreased by the addition of proteins that bind single-stranded DNA. Theaddition of E. coli single-strand binding protein (SSB) stimulatesSequenase-catalyzed HRCA, while phage T4 gene-32 protein stimulates Ventexo(−) catalyzed HRCA. In contrast, the Bst polymerase does not requiresuch ssingle-strand binding proteins for maximal activity.

[0306] If matrix-associated, the DNA product remains bound at the siteof synthesis, where it may be tagged, condensed, and imaged as a pointlight source. Linear oligonucleotide probes bound covalently on glasssurfaces can generate RCA signals, whose color indicates the allelestatus of the target, depending on the outcome of specific,target-directed ligation events. Single molecule counting by RCAprovides a powerful mutation detection method for studies usingoligonucleotide arrays, and is particularly amenable for the analysis ofrare somatic mutations.

[0307] In a further embodiment, the combined M-FISH/nucleic acidamplification methods of the invention (especially RCA/HRCA) may be usedto detect circularizable oligonucleotides, called “padlock probes”(Landegren, U. et al., Methods 9:84-90 (1996); Landegren, U. et al., AnnMed. 29:585-590 (1997); (Nilsson, M. et al., Science 265:2085-2088(1994); Nilsson, M. et al., Nat. Genet. 16: 252-255 (1997), all hereinincorporated by reference) bound to single copy genes in cytologicalpreparations.

[0308] Multiple applications exist for the above-described gap-fillreaction, in which target-complementary sequences are incorporated intocircles by copying and covalent closure. In principle, any DNA sequencethus captured into a circular DNA may be amplified by RCA or HRCA. Thereaction may thus be advantageously used in situations where it isdesirable to interrogate the sequence incorporated into a padlock probeat some point after RCA. Longer sequences, such as microsatelliterepeats, should also be capable of being copied into circularizableprobes for amplification and analysis. Subsequent to such copying, sincethere is little likelihood that rolling circle amplification will modifythe number of repeats incorporated into a circularized probe (Fire, A.et al., Proc. Natl. Acad Sci. (USA) 92:4641-4645 (1995)), directmeasurement of repeat size would be feasible.

[0309] In particular, any of three alternative HRCAIRCA strategies forallele discrimination utilizing ligation of circularizable DNA probesand rolling circle replication—“gap-probe RCA,” “polymerase-mediatedgap-fill RCA,” and “ligase-mediated extension of immobilized probeRCA”—may be employed in concert with the M-FISH methods of the presentinvention. The polymerase mediated “gap-fill” reaction is preferred overthe “gap probe” ligation reaction for solution studies of complexgenomes. This is because the gap oligonucleotides, which cannot bewashed away in a solution assay, are preferably used at relatively highconcentrations for the ligation step, and may therefore interfere withthe HRCA reaction, inducing the formation of amplicon artefacts. Incontrast, the gap ligation reaction is considered preferable for alleleanalysis of DNA in cytological specimens. Here, the specificity ofligation should be enhanced relative to probes without a gap becausethree different sequence recognition events and two independent ligationevents must occur before “padlock” closure. The third assay method,ligase-mediated extension of an oligonucleotide linked to a solidsurface, provides a totally novel approach to quantify individualhybridization/ligation events and to score rare somatic mutations.

[0310] In a highly preferred embodiment of HRCA/RCA, the DNA generatedby RCA is labeled with combinatorially labeled fluorescentDNP-oligonucleotide tags that hybridize at multiple sites in the tandemDNA sequence. The “decorated” DNA, labeled by specific encoding tags, isthen condensed into a small object by cross-linking with a multivalentantibody (e.g., anti-DNP IgM). The wild-type specific primer generatesRCA products which can hybridize to fluorescein-labeledDNP-oligonucleotide tags, while the mutant RCA products hybridize toCy3-labeled DNP-oligonucleotides. The process of Condensation ofAmplified Circles after Hybridization of Encoding Tags is termed“CACHET.” The use of CACHET, in concert with the M-FISH methods of thepresent invention is particularly preferred.

EXAMPLE 4

[0311] Use of M-FISH Analysis to Evaluate Telomere Integrity

[0312] Conventional analyses of chromosomes using, for example, Giemsabanding of metaphase chromosomes have several salient deficiencies. Inparticular, karyotypic changes involving chromosome fragments that havevery similar banding patterns can be impossible to resolve using thismethod (Saccone, E. et al., Proc. Natl. Acad. Sci. (USA) 89:4913-4917(1992)). Such a finding is particularly true of the telomeric bandswhich are virtually all Giemsa negative and which are also the mostgene-rich bands within the genome. The correct identification oftelomeric regions is very important because telomeres may be involved inbreakage and healing events that can result in terminal deletion, geneamplification or cryptic translocations.

[0313] Indeed, there is an increasing number of clinical cases that havebeen found to be the result of cryptic translocations such as hemoglobinH (Lamb, J et al., Lancet 2:819-824 (1989)), Cri-du-Chat (Overhauser, J.et al., Amer. J. Hum. Genet. 45:296-303 (1989)), Wolf-Hirschhorn(Altherr, M. R. et al, Amer. J. Hum. Genet. 49:1235-12342 (1991)), andMiller-Dieker lissencephaly syndrome (Kuwano, A. et al., Amer. J. Hum.Genet. 49:707-714 (1991)). In addition, cryptic translocations occur inup to 6% of the population with mild to moderate mental retardation whohave no detectable chromosomal changes upon karyotyping. Many of thesegenetic changes go undetected using standard cytogenetic analysis.

[0314] As indicated above, the present invention provides an ability tosimultaneously identify the twenty-four different human chromosomes in ametaphase spread by hybridizing a complete set of chromosome-specificDNA probes, each labeled with a different combination of dyes. Oneaspect of the present invention is the recognition that, through the useof telomere-specific probes, the methods of the present invention may beused to identify translocations that would be non-identifiable (i.e.,cryptic) through the use of conventional methods. As such, the presentinvention provides, for the first time, a simple screening test toassess the integrity of telomeric regions using a single hybridizationreaction. Such multiplex hybridization assays significantly improve theability to detect terminal deletions and cryptic chromosomalrearrangements, and thereby facilitate the identification of structuralabnormalities that elude detection with conventional cytogenetic bandingor multicolor whole-chromosome painting methods. Thus, this aspect ofthe present invention extends prior efforts in both karyotypingtechnology (see, e.g., Speicher, M. R. et al., Nature Genet 12:368-375(1996); Speicher, M. R. et al., Bioimaging 4:52-64 (1996); Schrock, E.et al., Science 273:494-497 (1996)) and telomere integrity assaytechnology (see, e.g., Ledbetter, D. H., Amer. J. Hum. Genet. 51:451-456(1992)); all herein incorporated by reference).

[0315] To illustrate this capacity of the invention, a set of YAC clonescontaining human DNA sequences specific for the sub-telomeric region ofeach chromosome arm is assembled. This probe set employed amicrodissected probe for the short arm of the Y chromosome (Guan, X. Y.,Nature Genet. 12:10-11 (1996), herein incorporated by reference) becauseno suitable sub-telomeric YAC for the short arm could be identified.Similarly, because of extensive cross-hybridization, the probe set didnot include probes for the p arms of the acrocentric chromosomes 13, 14,15, 21 and 22. The probes are combinatorially labeled such that thetelomeric regions of these chromosomes are visualized in differentpseudocolors based upon their unique fluorophore composition.

[0316] The p arm and q arm telomere proximal probes for each chromosomeare labeled with the same “color code,” for example both chromosome 1telomeres are labeled in color “a,” while the chromosome 2 telomeres arelabeled in color “b,” etc. Thus, any cytogenetically cryptic chromosometranslocation occurring on a non-acrocentric chromosome are visualizedin the metaphase spreads as a chromosome harboring 2 colors rather thana single color per chromosome. Although the 24 color telomere integrityassays utilize a complex probe mixture, such probe cocktails can behybridized with high reproducibility. Such hybridization is visualized,as described above, with, for example, an epifluorescence microscopeequipped with an appropriate filter set and a cooled CCD-camera, etc.The fluors and high contrast filters are the same as those describedabove. In brief, fluorescein (FLU), Cy3, and Cy5 are linked to dUTP fordirect labelling; Cy3.5 and Cy7 are available as avidin orantidigoxigenin conjugates for secondary detection of biotin- ordigoxigenin-labelled probes. For each probe, 1-3 separate nicktranslations are performed.

[0317] The telomeric probe set may be composed of a mixture of YACs,Half-Yacs (Vocero-Akbani, A. et al., Genomics 36:492-506 (1996); Macini,R. A. et al., Genomic Res. 5:225-232 (1995)), and CEPH-YACs (Chumakov,I. M. et al., Nature 377:175-297 (1995)), chosen to contain the mosttelomeric genetic markers. Half-YACs are generated from specialized YAClibraries enriched for telomeric sequences. The term “half-YACs” refersto the fact that one of the vector telomere sequences of the molecule isprovided by human DNA rather than yeast DNA. Half-YACs containsub-telomere repeats that are known to reside on different chromosomesthus, in some instances, they hybridize to multiple telomeres. However,specific M-FISH signals can be obtained from half-YACs, for examplethrough the use of DNA amplification (using, for example, PCR with Aluprimers to reduce the relative representation of repeat sequences);addition of Cot-1 DNA to the hybridization cocktail will suppresshybridization of any residual repetitive sequences. Suitable YACS,half-YACs and/or CePH-YACs are described by Vocero-Akhani, A. et al.(Genomics 36:492-506 (1996)), by the National Intitutes of Health andInstitute of Molecular Medicine Collaboration (Nature Genet 14:86-89(1996)) and by Bray-Ward, P. et al. (Genomics 36:1-14 (1996)); all ofwhich references are herein incorporated by reference. The fluors usedto label telomeric regions are shown in Table 4. In Table 4, thereferences are (1) Vocero-Akbani, A. et al., Genomics 36:492-506 (1996);(2) National Intitutes of Health and Institute of Molecular MedicineCollaboration (Nature Genet 14:86-89 (1996)); (3) Bray-Ward, P. et al.(Genomics 36:1-14 (1996)); (4) Bray-Ward, P. et al. (Genomics 36:104-111(1996)); (5)Guan, X. Y., Nature Genet. 12:10-11 (1996)), all hereinincorporated by reference. TABLE 4 Evidence for Most Distal TelomericInsert STS on Localization/ FISH Library Size Integrated Flpter TelomereProbe Label Source (kb) Mapl (min-max) Reference 1p TYAC179 Flu/Cy3HDK-TYACL 200 sJCW15 Half-YAC 1 (HTY3222) 1q yRM2123 Flu/Cy3 HR-TYACL270 Half-YAC 2 2p TYAC47 CY5/Cy7 HDK-TYACL 350 sAVA22 Half-YAC 1(HTY3047) CEPH-L 1350 D2S359 0.01-0.06 3 695h7 CEPH-L 1280 D2S3040.01-0.06 3 955c11 2q yRM2112 CY5/Cy7 HR-TYACL 240 sSMMI Half-YAK 2TYAC75 HDK-TYACL 75 Half-YAC 1 933c7 CEPH-L 1690 D2S159 0.93-0.95 3 3p852b3 Flu/Cy7 CEPH-L 1100 D3S1270 0.00-0.05 3 3q TYAC162 Flu/Cy7HDK-TYACL 250 Sava54 Half-YAC 1 (HTY3205) 4p 764h1 Cy3.5/Cy5/Cy7 CEPH-L400 3104 3 4q 946a3 Cy3.5/Cy5/Cy7 CEPH-L 1120 d4s2930 0.97-1.00 3 5p789d2 Cy3/Cy7 CEPH-L 1630 D5S676 3 5q TYAC139 Cy3/Cy7 HDK-TYACL 75Half-YAC 1 (HTY3182) CEPH-L 945 D5S498 0.91-1.00 3 887f10 6p 870d6Flu/Cy3/Cy7 CEPH-L 1260 D6S344 0.00 4 6q 820f9 Flu/Cy3/Cy7 CEPH-L 1050D6S281 0.92-0.95 3 7p 626g11 Cy3.5/Cy7 CEPH-L 290 D7S531 3 7q yRM2000Cy3.5/Cy7 HR-TYACL 240 Half-YAC 2 TYAC109 HDK-TYACL Half-YAC 1 (HTY3152)8p yRM2205 Cy3/Cy5 HR-TYACL 250 D8S264 Half-YAC 2 931b2 CEPH-L 0.00-0.043 8q yRM2053 Cy3/Cy5 HR-TYACL 170 2058VI Half-YAC 2 TYAC48 HDK-TYACL 200Half-YAC I (HTY3048) 9p 822e7 Flu/Cy3/Cy5 CEPH-L 1640 D9S178 3 9q 908a4Flu/Cy3/Cy5 CEPH-L 900 D9S1872 0.86-0.91 3 10p TYAC95 Flu/Cy5 HDK-TYACLHalf-YAC 1 (HTY3138) 10q TYAC93 Flu/Cy5 HDK-TYACL 250 Half-YAC 1(HTY3136) 11p yRM2209 Cy3.5/Cy5 HR-TYACL 125 D11S2071 Half-YAC 2 11q790g9 Cy3.5/Cy5 CEPH-L 1060 D11S968 0.95-1.00 3 12p 922c8 Cy3/Cy3.5CEPH-L 1390 D12S1455 0.01-0.05 3 12q TYAC175 Cy3/Cy3.5 HR-TYACL 250Half-YAC 1 (HTY3218) 13q yRM2067 Cy3/Cy3.5/Cy5 HR-TYACL Half-YAC 2 14qyRM2006 Cy3.5 HR-TYACL 200 Half-YAC 2 15q 877h4 Flu/Cy3.5/Cy7 CEPH-L 610WI-5214 3 16p 927a8 Cy7 CEPH-L 120 D16S423 0.02-0.06 3 16q TYAC135 Cy7HDK-TYACL 100 Sava47 Half-YAC 1 (HTY3178) 17P 898a10 Flu CEPH-L 570wi-5436 3 17q 965f1 Flu CEPH-L 1470 CHCLGATA4 3 FOR 18p yRM2102Flu/Cy3/Cy3.5 HR-TYACL 220 Half-YAC 2 18q yRM2050 Flu/Cy3/Cy3.5 HR-TYACL290 Half-YAC 2 19p 872g3 Cy3 CEPH-L 590 WI-9127 3 19q 926g5 Cy3 CEPH-L1440 D19S887 3 20p 761d12 Flu/Cy3 CEPH-L 1330 AFMA049Y 3 D1 20q 793a9Flu/Cy3 CEPH-L 1620 D20S173 3 21q yRM2208 Flu/Cy3.5/Cy5 HR-TYACL 290Half-YAC 2 22q TYAC104 Cy3/Cy3.5/Cy7 HDK-TYACL 75 Sava39 Half-YAC 1(HTY31476) Xp 827e10 Cy5 CEPH-L 920 wi-3553 3 Xq 883h10 Cy5 CEPH-L 1240wi-7878 3 Yq MDP Flu/Cy5/Cy7 NIH 5

[0318] In order to test the reproducibility of specific signals withhalf YAC probes, each is carefully tested in single color experiments onnormal metaphase spreads from at least five different donors. Thesestudies permit the assessment of polymorphisms as well as the frequencyor cross-hybridizations to other sub-telomeric repeats. Suitablehalf-YACs perform very reproducibly; in a few cases where occasionalcross-hybridization occur, the nonspecific signals usually had very lowfluorescence intensities and thus can be easily discriminated from areal signal.

[0319] CEPH-YACs were selected to contain the most telomeric geneticmarkers. Because the distance from the true telomeric end of thechromosome is unknown, these probes were tested by FISH for their distalband location. For six telomeric regions (2p, 2q, 5q, 7q, 8p, 8q) thehybridization of a single sub-telomeric YAC yielded low fluorescenceintensities. Therefore two (5q, 7q, 8p, 8q) or three (2p, 2q)subtelomeric YACs were pooled in order to increase the signal.Alu-fingerprinting analysis was used to confirm that the YACs overlap.

[0320] A modified version of the previously published M-FISH softwarefor whole chromosome painting probes was used for probe discrimination(Speicher, M. R. et al., Bioimaging 4:52-64 (1996); Schrock, E. et al.,Science 273:494-497 (1996)); Yale University). In brief, to correctimage shifts caused by optical and mechanical imperfections, wholechromosome painting probes, one for each fluor used in the experiment,are hybridized simultaneously with the pool of sub-telomeric YACs. Pixelshift correction of the chromosome painting probes is done as describedabove, however the chromosome paints are not shown on the final imagesto facilitate the color discrimination of the sub-telomeric probes.Instead of calculating a specific threshold for each fluor, based on thewhole chromosome paint signals, the terminal regions of the chromosomesare specifically analyzed. Chromosome ends are identified andsegmentation masks with different thresholds are calculated and checkedfor regions with increased fluorescence intensities. This is necessarybecause the simultaneous hybridization of multiple small region-specificprobes results in signals that are often in different focal planes. Inthe absence of Z-axis optical sectioning and image merging, this resultsin some YAC probes giving significantly reduced fluorescence intensityvalues. If a specific telomeric region is not labeled with a particularfluor, no increased fluorescence intensity peaks is observed in at leastone of the segmentation masks. The calculated fluor segmentation maskswere not overlayed with the DAPI segmentation mask that delineates thechromosomal boundaries (since some of the telomeric FISH signals can lieoutside the DAPI segment mask). Individual YAC-clones first are assigneddistinct gray values depending on the boolean signature of each probe orthe combination of fluors used to label it (Speicher, M. R. et al.,Bioimaging. 4:52-64 (1996); Schrock, E. et al., Science 273:494-497(1996)). A look-up-table then is used to assign each DNA target apsuedocolor depending on this gray value. Finally, this pseudocoloredimage was overlayed onto the DAPI-stained chromosome image that wasassigned a light blue color.

[0321] In order to optimize the experimental parameters for hybridizingsuch a large number of combinatorially labeled YAC clonessimultaneously, the multiplex telomere probe set is hybridized to normalmetaphase spreads from peripheral blood lymphocytes. Similar to theresults described above obtained with whole chromosome painting probes,YAC probes that are labeled with equal amounts of different fluors donot always give equal signal intensities for each fluor. To diminishsignal intensity differentials, probe concentrations for thehybridization mix and a reliable combinatorial labeling scheme aretherefore established by control experiments. YACs yielding largefluorescence signals are preferentially labeled with three differentfluors while YACs yielding rather weak signals were labeled with onefluor only.

[0322] Using such probes, a 24 color telomere integrity assay isconducted on karyotypes of 10 normal male and female donors. Noindication of polymorphisms are detected thereby suggesting that cryptictranslocations are extremely infrequent in normal populations. A typicalmetaphase spread is observed (FIG. 5A), and a normal karyotype based onthe boolean signature of our subtelomere probes is attained as expected(FIG. 5B).

[0323] In the process of testing the 24 color telomere integrity assay,the probe set is hybridized on metaphase spreads from a patient with amyeloproliferative disorder. Karyotyping using G-bands reveals trisomy 8as the only detectable cytogenetic change. This is verified by M-FISHusing chromosome specific painting probes. The telomere integrity assay,however, yields an intriguing hybridization pattern on the chromosomes8. The trisomy 8 is verified in all 10 metaphase spreads analyzed.However, on 8 of these 10 metaphase spreads a split telomere signal isobserved on two chromosomes 8, indicating an inversion in this region(FIG. 6). This analysis demonstrates the power of this new technique todetect structural abnormalities that are undetectable by standardcytogenetic methods or 24-color techniques using chromosome specificpainting probes.

[0324] Cases where the application of the telomere-integrity assay maybe very rewarding include evaluating karyotypes of patients with mentalretardation, a combination of mental retardation and dysmorphicfeatures, and cancer cells that are known to have a high genomicinstability. The latter cells should be highly predisposed tosub-telomeric translocation events. Screening for cryptic translocationscan not be done efficiently with chromosome specific painting probesbecause they were not designed to detect subtle deletion or rearrangmentevents.

[0325] In a further embodiment of the above methods, thetelomere-specific probe sets can be improved using PCR-assistedchromatography (Craig, J. et al., Hum. Genet 100:472-476 (1997), hereinincorporated by reference). This tool removes repetitive DNA, includingthe polymorphic repetitive sequences from half-YACs or other M-FISHprobes, and thereby facilitates the generation of probes that willhybridize specifically in the absence of Cot-1 suppressor DNA.

[0326] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A set of combinatorially labeled oligonucleotideprobes comprised of a first and a second subset of probes, wherein: (A)each member of said first subset of probes comprises a plurality of anoligonucleotide: (i) being linked or coupled to a predetermined labeldistinguishable from the label of any other member of said first orsecond subsets of probes, and (ii) being capable of specificallyhybridizing with one predetermined autosomal or sex chromosome of ahuman karyotype; said first subset of probes set having sufficientmembers to be capable of specifically hybridizing each autosomal or sexchromosome of said human karyotype to at least one member; and (B) eachmember of said second subset of probes comprises a plurality of anoligonucleotide: (i) being linked or coupled to a predetermined labeldistinguishable from the label of any other member of said first orsecond subset, and (ii) being capable of specifically hybridizing withone extra-chromosomal polynucleotide copy of a predetermined region ofan autosomal or sex chromosome of said human karyotype.
 2. The set ofcombinatorially labeled oligonucleotide probes of claim 1, wherein saidextra-chromosomal polynucleotide copy of a predetermined region of anautosomal or sex chromosome of said human karyotype is an RNA molecule.3. The set of combinatorially labeled oligonucleotide probes of claim 1,wherein said extra-chromosomal polynucleotide copy of a predeterminedregion of an autosomal or sex chromosome of said human karyotype is aDNA molecule.
 4. The set of combinatorially labeled oligonucleotideprobes of claim 1, wherein said set contains at least one member probespecific for a sub-chromosomal fragment of an autosomal or sexchromosome of said human karyotype.
 5. A set of combinatorially labeledoligonucleotide probes comprised of a first subset of genotypic probesand a second subset of phenotypic probes, wherein: (A) each member ofsaid first subset of genotypic probes comprises a plurality of anoligonucleotide: (i) being linked or coupled to a predetermined labeldistinguishable from the label of any other member of said first orsecond subsets of probes, and (ii) being capable of specificallyhybridizing with a region of a nucleic acid of a preselected bacterium,virus or lower eukaryote; said first subset of probes set havingsufficient members to be capable of distinguishing said preselectedbacterium, virus, or lower eukaryote from other bacteria, viruses, orlower eukaryotes; and (B) each member of said second subset ofphenotypic probes comprises a plurality of an oligonucleotide: (i) beinglinked or coupled to a predetermined label distinguishable from thelabel of any other member of said first or second subset, and (ii) beingcapable of specifically hybridizing with a predetermined polynucleotideregion of said chromosome of said preselected bacterium, virus, or lowereukaryote, or an extra-chromosomal copy thereof so as to permit thedetermination of whether said preselected bacterium, virus, or lowereukaryote exhibits a preselected phenotype.
 6. The set ofcombinatorially labeled oligonucleotide probes of claim 5, wherein saidextra-chromosomal polynucleotide copy of a predetermined region of saidchromosome is an RNA molecule.
 7. The set of combinatorially labeledoligonucleotide probes of claim 5, wherein said extra-chromosomalpolynucleotide copy of a predetermined region of said chromosome is aDNA molecule.
 8. The set of combinatorially labeled oligonucleotideprobes of claim 5, wherein said predetermined region of said chromosomeof said bacterium, virus or lower eukaryote encodes an antibioticresistance deterninant.
 9. The set of combinatorially labeledoligonucleotide probes of claim 5, wherein said predetermined region ofsaid chromosome of said bacterium, virus or lower eukaryote encodes atoxin.
 10. The set of combinatorially labeled oligonucleotide probes ofclaim 5, wherein said predetermined region of said chromosome of saidbacterium, virus or lower eukaryote encodes a protein that isdeterminative of the species of said bacterium, virus or lowereukaryote.
 11. The set of combinatorially labeled oligonucleotide probesof claim 10, wherein said predetermined region of said chromosome ofsaid bacterium, virus or lower eukaryote encodes a protein that isdeterminative of the subspecies of said species of bacterium, virus orlower eukaryote.
 12. The set of combinatorially labeled oligonucleotideprobes of any of claims 3 and 7, wherein said DNA molecule is producedthrough in situ nucleic acid amplification.
 13. The set ofcombinatorially labeled oligonucleotide probes of any of claims 3 and 7,wherein said in situ nucleic acid amplification is an in situ polymerasechain reaction.
 14. The set of combinatorially labeled oligonucleotideprobes of any of claims 3 and 7, wherein said in situ nucleic acidamplification is an in situ rolling circle amplification reaction. , 15.The set of combinatorially labeled oligonucleotide probes of any ofclaims 3 and 7, wherein said probes contain nucleotide residues that arelabeled with a biotin moiety.
 16. The set of combinatorially labeledoligonucleotide probes of claim 15, wherein said probes are additionallylabeled with a labeled biotin-binding ligand.
 17. The set ofcombinatorially labeled oligonucleotide probes of claim 16, wherein atleast one of said probes is labeled with a biotin-binding ligand thatcomprises one or more fluorophores.
 18. The set of combinatoriallylabeled oligonucleotide probes of claim 17, wherein at least one of saidfluorophores is selected from the group consisting of the fluorophoresFITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
 19. The set of combinatoriallylabeled oligonucleotide probes of claim 17, wherein at least one of saidprobes is labeled with a biotin-binding ligand that comprises more thanone fluorophore.
 20. The set of combinatorially labeled oligonucleotideprobes of claim 19, wherein one of the fluorophores of saidbiotin-binding ligand that comprises more than one fluorophore isselected from said group of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5and Cy7.
 21. The set of combinatorially-labeled oligonucleotide probesof claim 20, wherein all of the fluorophores of said biotin-bindingligand that comprises more than one fluorophore are selected from saidgroup of fluorophores FITC Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
 22. The setof combinatorially labeled oligonucleotide probes of claim 17, whereinat least one member of said set is labeled with more than onefluorophore selected from the group consisting of the fluorophores FITC,Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of said set islabeled with at least one fluorophore selected from said fluorophoregroup.
 23. The set of combinatorially labeled oligonucleotide probes ofclaim 22, wherein at least one member of said set is labeled with twofluorophores, each selected from the group consisting of thefluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein eachmember of said set is labeled with at least one fluorophore selectedfrom said fluorophore group.
 24. The set of combinatorially labeledoligonucleotide probes of claim 22, wherein at least one member of saidset is labeled with three fluorophores, each selected from the groupconsisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, andwherein each member of said set is labeled with at least one fluorophoreselected from said fluorophore group.
 25. A method of simultaneouslyidentifying and distinguishing the individual autosomal and sexchromosomes of a human karyotype which comprises the steps: (I)contacting a preparation of said chromosomes, in single-stranded form,under conditions sufficient to permit nucleic acid hybridization tooccur with a set of combinatorially labeled oligonucleotide probescomprised of a first and a second subset of probes, wherein: (A) eachmember of said first subset of probes comprises a plurality of anoligonucleotide: (i) being linked or coupled to a predetermined labeldistinguishable from the label of any other member of said first orsecond subsets of probes, and (ii) being capable of specificallyhybridizing with one predetermined autosomal or sex chromosome of ahuman karyotype; said first subset of probes set having sufficientmembers to be capable of specifically hybridizing each autosomal or sexchromosome of said human karyotype to at least one member; and (B) eachmember of said second subset of probes comprises a plurality of anoligonucleotide: (i) being linked or coupled to a predetermined labeldistinguishable from the label of any other member of said first orsecond subset, and (ii) being capable of specifically hybridizing withan a predetermined extra-chromosomal polynucleotide copy of a region ofan autosomal or sex chromosome of said human karyotype. (II) for eachchromosome of said preparation hybridized to a member of said firstsubset of probes, detecting and identifying the predetermined label ofthat member and correlating the identity of the label of that memberwith the identity of the autosomal or sex chromosome of said humankaryotype with which that member specifically hybridizes, to therebyidentify the chromosome hybridized to said member; (III) repeating step(II) until each autosomal and sex chromosome of said human karyotype hasbeen identified in said preparation (IV) for each member of said secondsubset of probes hybridized to a predetermined extra-chromosomalpolynucleotide copy of a region of an autosomal or sex chromosomedetecting and identifying the predetermined label of that member andcorrelating the identity of the label of that member with the identityof the region of the autosomal or sex chromosome of said human karyotypewith which that member specifically hybridizes, to thereby identify theregion of said autosomal or sex chromosome hybridized to said member;(V) repeating step (IV) for each member of said second subset of probes.26. The method of claim 25, wherein said extra-chromosomalpolynucleotide copy of said predetermined region of an autosomal or sexchromosome of said human karyotype is an RNA molecule.
 27. The method ofclaim 25, wherein said extra-chromosomal polynucleotide copy of saidpredetermined region of an autosomal or sex chromosome of said humankaryotype is a DNA molecule.
 28. The method of claim 25, wherein saidset of oligonucleotide probes contains at least one member probespecific for a sub-chromosomal fragment of an autosomal or sexchromosome of said human karyotype.
 29. A method of simultaneouslyidentifying and distinguishing a preselected bacterium, virus, or lowereukaryote from other bacteria, viruses or lower eukaryotes that may bepresent in a sample which comprises the steps: (I) contacting apreparation suspected to contain said preselected bacterium, virus, orlower eukaryote, under conditions sufficient to permit in situ nucleicacid hybridization to occur, with a set of combinatorially labeledoligonucleotide probes comprised of a first subset of genotypic probesand a second subset of phenotypic probes, wherein: (A) each member ofsaid first subset of genotypic probes comprises a plurality of anoligonucleotide: (i) being linked or coupled to a predetermined labeldistinguishable from the label of any other member of said first orsecond subsets of probes, and (ii) being capable of specificallyhybridizing with a region of a nucleic acid of said preselectedbacterium, virus or lower eukaryote; said first subset of probes sethaving sufficient members to be capable of distinguishing saidpreselected bacterium, virus, or lower eukaryote from other bacteria,viruses, or lower eukaryotes present in said preparation; and (B) eachmember of said second subset of probes comprises a plurality of anoligonucleotide: (i) being linked or coupled -to a predetermined labeldistinguishable from the label of any other member of said first orsecond subset, and (ii) being capable of specifically hybridizing with apredetermined polynucleotide region of said nucleic acid of saidpreselected bacterium, virus, or lower eukaryote, or anextra-chromosomal copy thereof so as to permit the determination ofwhether said preselected bacterium, virus, or lower eukaryote exhibits apreselected phenotype. (II) for each member of said first subset ofprobes hybridized to a region of a chromosome of a preselectedbacterium, virus or lower eukaryote, detecting and identifying thepredetermined label of that member and correlating the identity of thelabel of that member with the identity of the bacterium, virus or lowereukaryote with which that member specifically hybridizes, to therebyidentify said bacterium, virus or lower eukaryote hybridized to saidmember; (III) repeating step (II) for each member of said first subsetof probes; (IV) for each member of said second subset of probeshybridized to a predetermined polynucleotide region of said chromosomeof said preselected bacterium, virus, or lower eukaryote, or anextra-chromosomal copy thereof, detecting and identifying thepredetermined label of that member and correlating the identity of thelabel of that member with the identity of said predetermined region, tothereby identify the presence of said predetermined region on achromosome of said preselected bacteria, virus or lower eukaryote; (V)repeating step (IV) for each member of said second subset of probes. 30.The method of claim 29, wherein said extra-chromosomal polynucleotidecopy of said predetermined region of said chromosome of said bacterium,virus or lower eukaryote is an RNA molecule.
 31. The method of claim 29,wherein said extra-chromosomal polynucleotide copy of said predeterminedregion of said chromosome of said bacterium, virus or lower eukaryote isa DNA molecule.
 32. The method of claim 29, wherein said predeterminedregion of said chromosome of said bacterium, virus or lower eukaryoteencodes an antibiotic resistance determinant.
 33. The method of claim29, wherein said predetermined region of said chromosome of saidbacterium, virus or lower eukaryote encodes a toxin.
 34. The method ofclaim 29, wherein said predetermined region of said chromosome of saidbacterium, virus or lower eukaryote encodes a protein that isdeterminative of the species of said bacterium, virus or lowereukaryote.
 35. The method of claim 34, wherein said predetermined regionof said chromosome of said bacterium, virus or lower eukaryote encodes aprotein that is determinative of the subspecies of said species ofbacterium, virus or lower eukaryote.
 36. The method of any of claims 27and 31, wherein said in situ nucleic acid amplification is an in situpolymerase chain reaction.
 37. The method of claim 36, wherein said insitu nucleic acid amplification is an in situ rolling circleamplification reaction.
 38. The method of claim 36, wherein said probescontain nucleotide residues that are labeled with a biotin moiety. 39.The method of claim 36, wherein said probes are additionally labeledwith a labeled biotin-binding ligand.
 40. The method of claim 39,wherein at least one of said probes is labeled with a biotin-bindingligand that comprises one or more fluorophores.
 41. The method of claim40, wherein at least one of said fluorophores is selected from the groupconsisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. 42.The method of claim 40, wherein at least one of said probes is labeledwith a biotin-binding ligand that comprises more than one fluorophore.43. The method of claim 42, wherein one of the fluorophores of saidbiotin-binding ligand that comprises more than one fluorophore isselected from said group of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5and Cy7.
 44. The method of claim 43, wherein all of the fluorophores ofsaid biotin-binding ligand that comprises more than one fluorophore areselected from said group of fluorophores FlTC, Cy3, Cy3.5, Cy5, Cy5.5and Cy7.
 45. The method of claim 40, wherein at least one member of saidset is labeled with more than one fluorophore selected from the groupconsisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, andwherein each member of said set is labeled with at least one fluorophoreselected from said fluorophore group.
 46. The method of claim 45,wherein at least one member of said set is labeled with twofluorophores, each selected from the group consisting of thefluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein eachmember of said set is labeled with at least one fluorophore selectedfrom said fluorophore group.
 47. The method of claim 45, wherein atleast one member of said set is labeled with three fluorophores, eachselected from the group consisting of the fluorophores FITC, Cy3, Cy3.5,Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled withat least one fluorophore selected from said fluorophore group.
 48. Themethod of any of claims 25 and 29, wherein an optical optical comb isemployed in step (b) to detect the predetermined label of saidhybridized probe member.
 49. The method of claims 25 and 29, wherein anoptical filter is employed in step (b) to detect the predetermined labelof said hybridized probe member.
 50. The method of claim 49, wherein aplurality of optical filters is sequentially employed in step (b) todetect the predetermined label of said hybridized probe member.
 51. Themethod of claim 48, wherein at least one member of said set is labeledwith more than one fluorophore selected from the group consisting of thefluorophores FlTC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein eachmember of said set is labeled with at least one fluorophore selectedfrom said fluorophore group.
 52. The method of claim 49, wherein atleast one member of said set is labeled with two fluorophores, eachselected from the group consisting of the fluorophores FITC, Cy3, Cy3.5,Cy5, Cy5.5 and Cy7, and wherein each member of said set is labeled withat least one fluorophore selected from said fluorophore group.
 53. Themethod of claim 49, wherein at least one member of said set is labeledwith three fluorophores, each selected from the group consisting of thefluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein eachmember of said set is labeled with at least one fluorophore selectedfrom said fluorophore group.
 54. A set of combinatorially labeledoligonucleotide probes, each member thereof: (i) having a predeterminedlabel distinguishable from the label of any other member of said set,and (ii) being capable of specifically hybridizing with a telomericregion of one predetermined autosomal or sex chromosome of a humankaryotype; said set having sufficient members to be capable ofspecifically hybridizing each autosomal or sex chromosome of said humankaryotype to at least one member.
 55. The set of combinatorially labeledoligonucleotide probes of claim 54, wherein at least one of said labelsis a fluorophore selected from the group consisting of the fluorophoresFITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
 56. A method of simultaneouslyidentifying and distinguishing the individual autosomal and sexchromosomes of a human karyotype which comprises the steps: (a)contacting a preparation of said chromosomes, in single-stranded form,under conditions sufficient to permit nucleic acid hybridization tooccur with a set of combinatorially labeled oligonucleotide probes, eachmember thereof: (i) having a predetermined label distinguishable fromthe label of any other member of said set, and (ii) being capable ofspecifically hybridizing with a telomeric region of one predeterminedautosomal or sex chromosome of a human karyotype; said set havingsufficient members to be capable of specifically hybridizing eachautosomal or sex chromosome of said human karyotype to at least onemember; wherein said contacting thereby causes at least one of eachautosomal or sex chromosome of said preparation to become hybridized toat least one member of said set of probes; (b) for each chromosome ofsaid preparation hybridized to a member of said set of probes, detectingand identifying the predetermined label of that member and correlatingthe identity of the label of that member with the identity of theautosomal or sex chromosome of said human karyotype with which thatmember specifically hybridizes, to thereby identify the chromosomehybridized to said member; and (c) repeating step (b) until eachautosomal and sex chromosome of said human karyotype has been identifiedin said preparation.
 57. The method of claim 56, wherein at least one ofsaid labels is a fluorophore selected from the group consisting of thefluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.